Separation system, components of a separation system and methods of making and using them

ABSTRACT

Permeable polymeric monolithic materials are prepared in a column casing. In one embodiment, the permeable polymeric monolithic materials are polymerized while pressure is applied through a piston having a smooth piston head in contact with the polymerization mixture. The pressure eliminates wall effect and changes the structure in the column. Similarly, some columns that have a tendency to swell in the presence of aqueous solutions and pressurized while the solution is applied to prevent swelling and wall effect. This procedure also changes the structure in the column. The size of the separation effective openings can be controlled by the amount of the pressure and pores eliminated. Uniformity in the direction flow is improved by controlling polymerization with radiation rather than with conducted heat.

RELATED CASES

This application is a continuation of U.S. patent application Ser. No.10/607,080, filed Jun. 25, 2003, which is a continuation in part of U.S.patent application Ser. No. 10/180,350 filed Jun. 26, 2002 (now U.S.Pat. No. 6,749,749 issued Jun. 15, 2004), in the names of Shaofeng Xieand Robert W. Allington for SEPARATION SYSTEM, COMPONENTS OF ASEPARATION SYSTEM AND METHODS OF MAKING AND USING THEM.

BACKGROUND OF THE INVENTION

This invention relates to separation systems and their components andmore particularly to separation systems and components involvingmonolithic permeable polymeric materials.

Monolithic macroporous materials such as for example organic monolithicmacroporous polymeric materials and monolithic silica packings are knownas components for separation systems such as chromatographic orextraction systems. One class of such materials is formed as amonolithic macroporous polymer plug or solid support produced bypolymerizing one or more monomers in a polymerization mixture thatincludes at least a porogen. It is known for some polymerizationmixtures, to include other materials such as cross-linking agents,catalysts and small soluble polymers which can be dissolved afterpolymerization to control the porosity and pore size distribution.Moreover, the plug may be modified after being formed to add functionalgroups.

The plug or solid support is normally contained in a housing such as forexample a chromatographic column or a pressure vessel. The portion ofthe housing where the plug resides acts as a reactor. In one prior artprocess for making monolithic columns, the polymerization mixture may beadded to the column casing and polymerization initiated therein to forma macroporous polymeric plug or solid support within the walls of thecolumn.

There are wide applications of these plugs or solid supports includinggas, liquid and supercritical fluid chromatography, membranechromatography and filtration, solid phase extraction, catalyticreactors, solid phase synthesis and others. The efficiency of the columnor other container for the plug or solid support, the time required fora separation, and the reproducibility of the columns or other containerfor the plug or solid support are important commercial factors. Theefficiency of separation systems such as chromatographic columns withporous polymer in them is related to both the selectivity of the columnor other component containing the macroporous polymeric material and tozone spreading. Some of these factors are affected by moleculardiffusion and velocity of the mobile phase in the plug or solid supportduring a separation process.

The manner in which molecular diffusion and velocity of the mobile phaseaffects column efficiency can be in part explained by showing the effectof these factors on Height Equivalent to Theoretical Plates (HETP), theconventional designation of column efficiency. The van Deemter Equationshows the relationship between zone spreading, flow velocity anddiffusion in terms of H (HETP) as follows:

H=A+B/u+Cu with low H corresponding to high efficiency

U=Flow velocity of the mobile phase

A=Radial Eddy Diffusion coefficient

B=Longitudinal Molecular Diffusion coefficient

C=The mass transfer coefficient

Molecular diffusion depends on the diffusion of the molecules but not onthe packing of the bed. Eddy diffusion depends on the homogeneity of thepacking of the particles.

Zone spreading from mass transfer can be minimized by using non-porousparticles and porous particle with sizes smaller than 1.5 microns.However, packing with non-porous particles has extremely low surfacearea which is detrimental to the purification process (as opposed to theanalytical process) because the purification process requires highsample loading. The use of very small packed particles requires eitherhigh pressure which is difficult in most of the separation process usingcurrent instrumentation or low velocity which can increase the time fora given separation (sometimes expressed in H per minute).

The prior art separation systems that include as a component macroporouspolymeric monolithic plugs or solid supports use plugs or solid supportsformed from particles in the polymers that are larger than desired, lesshomogenous and include micropores. The large size of the particles andtheir lack of homogeneity result in a lack of homogeneity in the poresize distribution. The non-homogeneity of the pore sizes and largeamount of micropores in the prior art porous polymers contributesgreatly to the zone spreading as shown by the van Deemter Equation. Thelarge number of micropores contributes to zone spreading by capturingsample and retaining it for a time. This may be stated conventionally asthe non-equilibrium mass transfer in and out of the pores and betweenthe stationary phase and the mobile phase.

The prior art plugs or solid supports formed of porous polymers havelower homogeneity of pore size, less desirable surface features andvoids in their outer wall creating by wall effect and thus higher zonespreading and lower efficiency than desired in separation systems.

The prior art also fails to provide an adequate solution to a problemrelated to shrinkage that occurs during polymerization and shrinkagethat occurs after polymerization in some prior art porous polymers. Theproblem of shrinkage during polymerization occurs because monomers arerandomly dispersed in the polymerization solution and the polymersconsist of orderly structured monomers. Therefore, the volume of thepolymers in most of the polymerization is smaller than the volume of themixed monomers. The shrinkage happens during the polymerization in allof the above preparation processes. One of the problems with shrinkageafter polymerization occurs because of the incompatibility of a highlyhydrophilic polymer support with a highly hydrophilic aqueous mobilephase or other highly polar mobile phase such as for example, a solutionhaving less than 5-8 percent organic solvent content.

Shrinkage of the porous polymeric materials used in separation systemsand their components during polymerization results in irregular voids onthe surface of the porous polymers and irregularity of the pore sizeinside the polymer, which are detrimental to the column efficiency andthe reproducibility of the production process. One reason the columnefficiency is reduced by wall effect is that wall effect permits thesample to flow through the wall channels and bypass the separationmedia. One reason the reproducibility of the production process arereduced by wall effect is the degree of wall effect and location of thewall effect are unpredictable from column to column.

The columns with large channels in the prior art patents cited abovehave low surface area and capacity. The low capacities of the columnsare detrimental for purification process which requires high sampleloadings. In spite of much effort, time and expense in trying to solvethe problems of shrinkage, the prior art fails to show a solution toreduced capacity.

Because of the above phenomena and/or other deficiencies, the columnsprepared by the above methods have several disadvantages, such as forexample: (1) they provide columns with little more or less resolutionthan commercially available columns packed with beads; (2) theseparations obtained by these methods have little more or no betterresolution and speed than the conventional columns packed with eithersilica beads or polymer beads, particularly with respect to separationof large molecules; (3) the wide pore size distribution that resultsfrom stacking of the irregular particles with various shapes and sizeslowers the column efficiency; (4) the non-homogeneity of the pore sizesresulting from the non-homogeneity of the particle sizes and shapes inthe above materials contribute heavily to the zone spreading; (5) thelarge amount of micropores in the above materials also contributesgreatly to the zone spreading; and (6) shrinkage of the material used inthe columns reduces the efficiency of the columns. These problems limittheir use in high resolution chromatography.

U.S. Pat. No. 5,453,185 proposed a method of reducing the shrinkage byreducing the amount of monomers in the polymerization mixture usinginsoluble polymer to replace part of the monomers. This reduces theshrinkage but is detrimental to the capacity and retention capacityfactor of the columns which require high amount of functional monomers.There is nothing mentioned in these patents regarding the detrimentaleffect of shrinkage on resolution and the resulting irregular voids onthe surface of the porous polymer and irregularity of the pore sizeinside the polymer, which are detrimental to the column efficiency andthe reproducibility of the production process.

Prior art European patent 1,188,736 describes a method of making porouspoly(ethylene glycol methacrylate-co-ethylene glycol dimethacrylate) byin situ copolymerization of a monomer, a crosslinking agent, a porogenicsolvent and an initiator inside a polytetrafuoroethylene tube sealed atone end and open at the other end. The resulting column was used forgas-liquid chromatography. This prior art approach has the disadvantageof not resulting in materials having the characteristics desirable forthe practical uses at least partly because it uses polymerization in aplastic tube with an open end.

U.S. Pat. No. 2,889,632, 4,923,610 and 4,952,349 disclose a method ofmaking thin macroporous membranes within a sealed device containing twoplates and a separator. In this method the desired membrane support waspunched out of a thin layer of porous polymer sheet and modified to havedesired functional groups. The layers of porous sheets are held in asupport device for “membrane separation”. These patents extended themethod described in European patent 1,188,736 to prepare a porousmembrane and improve the technique for practical applications inmembrane separation. The resulting material is a macroporous membraneincluding pores from micropores of size less than 2 nanometers to largepores. The size of the particles of the polymer is less than 0.5micrometers. The separation mechanism of membrane separation isdifferent from that of conventional liquid chromatography.

This porous material has several disadvantages, such as for example: (1)the thinness of the membrane limits its retention factor; and (2) thepores formed by these particles are small and can not be used at highflow rate with liquid chromatography columns that have much longer bedlengths than the individual membrane thicknesses. The micropores andother trapping pores trap molecules that are to be separated andcontribute to zone spreading. The term “trapping pore” in thisspecification means pores that contribute to zone spreading such aspores ranging in size from slightly larger than the molecule beingseparated to 7 times the diameter of the pore being separated.

U.S. Pat. Nos. 5,334,310; 5,453,185 and 5,728,457 each disclose a methodof making macroporous poly(glycidyl methacrylateco-ethylene glycoldimethacrylate) polystyrene in situ within sealed columns. This methodextends the methods described in both the European patent 1,188,736 andU.S. Pat. Nos. 2,889,632, 4,923,610 and 4,952,349 for preparing liquidchromatography columns for the separation of proteins. U.S. Pat. Nos.5,334,310, 5,453,185 and 5,728,457 profess the intention of improvingthe column efficiency by removing the interstitial volume ofconventional packed columns having beads. The plugs formed according tothese patents have a pore size distribution that is controlled by thetype and amount of porogens, monomers and polymerization temperature.The macroporous polymers consist of interconnected aggregates ofparticles of various sizes which form large pore channels between theaggregates for the transport of the mobile phase. Among the aggregatesor clusters there exist small pores for separations. The small particlesare formed from tightly packed extremely small particles ca 100-300nanometers.

The materials made in accordance with these patents have a disadvantagein that the micropores within or between these particles physically trapthe sample molecules and degrade the separation. Although these patentsclaim that there are no interstitial spaces in the monolithic media asin the packed bed with beads, the large channels between the aggregatesand interconnected particles actually cause the same problem as theinterstitial spaces between the beads in conventional packed columnswith beads. The large channels formed from various size of aggregates orclusters are inhomogeneous and provide random interstitial spaces, evenwith narrow particle size distribution. Because of the randominterstitial spaces the column efficiency is poor.

U.S. Pat. Nos. 5,334,310, 5,453,185 and 5,728,457 disclose thepreparation of the separation media inside a column with cross sectionarea from square micrometers to square meters. The processes disclosedin these patents have some disadvantages. Some of the disadvantages weredisclosed by the inventors named in those patents in 1997 in Chemistryof Materials, 1997, 9, 1898.

One significant disadvantage is that larger diameter (26 mm I.D.)columns prepared from the above patented process have a pore sizedistribution is too irregular to be effective in chromatographyseparation. The irregular pore size distribution is caused by thedetrimental effect of polymerization exotherm, the heat isolating effectof the polymer, the inability of heat transfer, auto accelerateddecomposition of the initiator and concomitant rapid release of nitrogenby using azobisisobutyronitrile as initiator in a mold with 26 mmdiameter. It has been found that the temperature increase anddifferential across the column created by the polymerization exothermand heat transfer difficulties results in accelerated polymerization inlarge diameter molds such as for example molds having a diameter of morethan 15 mm and in a temperature gradient between the center of thecolumn and the exterior wall of the column which results ininhomogeneous pore structure. It was suggested in this article that theproblem might be reduced by slow addition of polymerization mixture.This helps to solve the problem partly but does not solve the problemcompletely. There is still a temperature gradient for the largerdiameter columns, which result in in-homogeneity of the pore sizedistribution.

This problem was also verified by theoretical calculations in thepublication of Analytical Chemistry, 2000, 72, 5693. This authorproposes a modular approach by stacking thin cylinders to constructlarge diameter columns for radial flow chromatography. However, sealingbetween the discs to form a continuous plug is difficult and timeconsuming.

U.S. Pat. Nos. 5,334,310, 5,453,185 and 5,728,457 disclose the materialof weak anion exchange and reversed phase columns. The weak anionexchanger prepared had low resolution, low capacity, low rigidity, slowseparation and very poor reproducibility. The reversed phase media hasvery little capacity, noh-ideal resolution, and very poorreproducibility. They can not be used in mobile phase with high watercontent such as less than 8% acetonitrile in water due to wallchanneling effect resulting from shrinkage of the very hydrophobic mediain this very polar mobile phase. This media is also compressed duringseparation and result in excess void volume in the head of the column.The above patents provide little guidance on how to prepare a weakcation exchanger, strong cation exchanger, strong anion exchanger,normal phase media and hydrophobic interaction media. These media basedon membrane, beads or gels are known. However, the preparation are doneby off-line and can not be used for in situ preparation of monolithiccolumns. The monolithic membrane prepared according to U.S. Pat. Nos.2,889,632, 4,923,610 and 4,952,349 has low capacity and resolution.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide an improvedseparating system having synergistic relationships with a polymer havingseparation-effective openings.

It is a further object of the invention to provide an improvedchromatographic column.

It is a still further object of the invention to provide an improvedapparatus for making a chromatographic column.

It is a further object of the invention to provide an improved methodfor forming chromatographic columns.

It is a still further object of the invention to provide an improvedpermeable monolithic medium.

It is a still further object of the invention to provide a permeablemonolithic column with improved resolution.

It is a still further object of the invention to provide a permeablemonolithic column with improved capacity.

It is a still further object of the invention to provide a column withimproved flow rate.

It is a still further object of the invention to provide a column withreduced tendency to swell when used with aqueous solvent.

It is a still further object of the invention to provide a column withimproved reproducibility.

It is a still further object of the invention to provide an improvedtechnique for the formation of permeable monolithic columns withcontrolled pore size selected for the purpose of improving capacity orresolution or flow rate.

It is a still further object of the invention to provide a method ofpreparing large diameter columns for preparative separations.

It is a still further object of the invention to provide a method ofpreparing polymeric materials including permeable polymer support usingirradiation methods.

It is a still further object of the invention to provide an improvedweak anion exchange column.

It is a still further object of the invention to provide an improvedreverse phase column.

It is a still further object of the invention to provide a highperformance strong anion exchange column.

It is a still further object of the invention to provide a highperformance weak cation exchange column.

It is a still further object of the invention to provide a highperformance strong cation exchange column.

It is a still further object of the invention to provide a highperformance normal phase column.

It is a still further object of the invention to provide a method ofavoiding a reduction in the quality of a separating medium caused byshrinkage during polymerization or swelling of the medium during washingor during separation.

It is a still further object of the invention to provide achromatographic system in which an array of columns, in which thecolumns are very close in characteristics, are operated together.

It is a still further object of the invention to provide a novel processfor making monolithic permeable solid support for applications inchromatography including liquid, gas and supercritical fluidchromatography, electrochromatography, catalytic reactor, filtration orothers requiring permeable polymer supports or solid permeable polymerholders adjacent or positioned at least partly horizontally from asample.

It is a further object of the invention to provide a novel highresolution media and novel method of obtaining it.

It is a further object of the invention to provide an improved permeablesolid support with homogeneous separation-effective opening sizedistribution resulting from more homogeneous size and shape of theinterconnected aggregated particles.

It is a further object of the invention to provide an improved permeablesolid support with less or no micropore.

It is a further object of the invention to provide an improved permeablesolid support with no voids on the wall of the polymeric support.

It is a further object of the invention to provide an method forimproving the capacity of monolithic chromatography media.

It is a still further object of the invention to provide a permeablemonolithic column with improved resolution.

It is a still further object of the invention to provide an improvedtechnique for the formation of permeable monolithic columns withcontrolled size separation-effective openings selected for the purposeof improving capacity or resolution or flow rate.

It is a still further object of the invention to provide a method ofpreparing large diameter columns for preparative separations.

It is a still further object of the invention to provide a highperformance catalytic reactor.

It is a still further object of the invention to provide a highperformance solid phase extraction bed.

It is a still further object of the invention to provide an improvedpermeable monolithic medium with covalently bonded particles having acontrolled minute thoroughly convoluted surface configuration but withfew or no micropores.

It is still a further object of the invention to provide a monolithicchromatographic bed with negligible nonuniformities due to thermaleffects during polymerization reaction.

It is a still further object of the invention to provide a novel methodfor controlling polymerization in a chromatographic column with relianceon conduction of heat into the column.

It is still a further object of the invention to control the rate ofpolymerization, and resulting thermal gradients, by means of controlledradiation impinging on the bed.

It is still a further object of the invention to control polymerizationwith a relatively safe radiation source such as those providing mediumenergy x-ray (e.g. below 200 kEV), UV or visible radiation.

It is a still further object of the invention to provide a permeable,high capacity, column with few or no micropores.

It is a still further object of the invention to provide a method ofpreparing columns with small diameters from nanometers to millimeters.

It is a still further object of the invention to provide a method ofpreparing chromatographically uniform columns with medium diameters upto 100 mm and large diameter columns up to 1000 mm.

In accordance with the above and further objects of the invention, apolymerization mixture is polymerized in place with a porogen or solventto form a polymer plug that has separation effective openings. In thisspecification, “separation-effective openings” means pores or channelsor other openings that play a role in separation processes such as forexample chromatography. Pores generally means openings in the particlesthat are substantially round and may be through pores passing throughparticles (through pores) or openings into the particles or in somecases, openings into or through aggregates of particles. By beingsubstantially round in cross-section, it is meant that the pores are notperfect circles and for example may be bounded by sectors of imperfectspheres with the pores being the open spaces between the adjacentspherical surfaces.

Some other terms are defined below as they are used in thisspecification. Separation factors includes those factors that effectretention and capacity or other factors that play a role in separationprocesses. The term “macroporous” in this specification is given itsusual meaning in referring to monolithic materials in separationsystems. Its usual meaning refers to pores or other voids betweenglobules of particles, which pores or other voids have a diameter ofover 50 nm. regardless of the length of an opening, rather than itsliteral connotation that would limit the openings to pores with asubstantially circular cross section and no cross sectional dimensionsubstantially longer than the other. The term “permeable” in thisspecification shall be interpreted in the same manner as “macroporous”with reference to monolithic materials in the separation arts but isused in preference to the term “macroporous” to distinguish materialshaving channels and other openings from those containing pores to avoidconfusion with the literal meaning of the term “macroporous”. In thisspecification the term “permeable non-porous” describes media havingopenings such as channels or the like but not containing pores asdefined above.

In one embodiment of this invention, shrinkage during polymerization iscompensated for and In another embodiment of this invention, swellingafter polymerization, which might otherwise later result in shrinkage isavoided. Shrinkage results in enlarged voids on the polymer surface andmay result in a lack of homogeneity of pore size distribution inside thepolymer. The voids are believed to be created by decreased volume oforderly structured polymer compared to the volume of monomers prior topolymerization when created during polymerization. The voids are mostlylocated in between the column wall and polymer due to the difference insurface free energy. The voids are probably occupied by the nitrogen gasgenerated by azobisisobutyronitrile (AIBN), which is a common initiatorfor the polymerization.

In a first embodiment, the compensation for shrinkage is accomplished byapplying sufficient pressure during polymerization to create uniformityin the distribution of separation-effective openings and to avoid walleffect voids. This pressure has been found to also control particle sizeand the nature and shape of the openings in the plug to some extent.Maintaining the column at atmospheric pressure during polymerization toaccommodate shrinkage does reliably prevent the formation of voids.Generally 250 psi pressure is used for convenience but higher and lowerpressures have been used successfully. The voids are removed when theplug stops shrinking when put under even modest amounts of pressure. Ina second embodiment, shrinkage that otherwise would occur afterpolymerization is avoided. For example, some plugs tend to expand whenexposed to some solutions such as organic solvent and then shrink latersuch as during a separating run in aqueous mobile phase, causing voids.In these embodiments, shrinkage is prevented by holding the column fromshrinkage when exposed to the solutions. The application of pressure isone method of preventing shrinkage during exposure to the aqueoussolutions. Other methods for compensated for shrinking and/or swelling,for reducing shrinking or for avoiding shrinking are also used asdescribed in greater detail below. It is believed that the externallyapplied pressure overcomes uneven forces internal to the reactingpolymerization mixture and between the polymerization mixture andinternal wall of the column to maintain homogeneous separation effectivefactors, separation-effective opening size and distribution and uniformcontinuous contact of the polymer to the internal wall of the column.

Surprisingly, some types of polymer plugs contain no pores if they aresubject to pressure during polymerization to compensate for shrinking orin the case of some reversed phase columns to compensate for shrinkagewhen exposed to hydrophillic solutions such as for example in theaqueous mobile phase Instead, they contain solid particles ca 2micrometers in diameter, covalently bonded together with relativelylarge flow channels between them (separation-effective openings). Thesurprising thing is that, although these particles have no pores, thechromatographic capacity of the plug is high. This is believed to happenbecause of the unexpected formation of ca 50-200 nm deep and widegrooves or corrugations and other odd surface features. A typicalparticle resembles a telescopic view of a very small asteroid.

The pressure applied during polymerization is selected in accordancewith the desired result and may be, for example, a linearly increasingpressure, a constant pressure or a step pressure gradient. In oneembodiment, separation-effective opening size is controlled by selectingthe type and proportion of porogen that generates the pores duringpolymerization and the porogen that must be washed out of the plug afterpolymerization. This proportion is selected by trial runs to obtain thedesired characteristic. The total amount of porogen is also selected.

In another embodiment, some plugs tends to expand when exposed to somesolutions such as organic washing solutions and then shrinks later suchas during a separating run in the aqueous mobile phase, creating voidsbetween column wall and polymer support and variations inseparation-effective opening size distribution. For example, somereverse phase plugs with separation-effective openings may shrink whenpolymerized others may not, and after polymerization, some of the plugsthat did not shrink during polymerization and some that did may shrinkif exposed to water or some other polar solutions. In this case, thecompensation for this shrinkage is the compression with a piston duringpolymerization and/or compression after polymerization during conditionsthat would normally cause shrinking equal or more than the shrinkagethat could happen during the separation run to force reordering orrepositioning or to compensate for the shrinking. In either case whereshrinkage is compensated for with pressure or where shrinkage isprevented to avoid causing voids, non-fluidic pressure such as with apiston is preferred rather than pressure with fluid. The word “pressure”in this specification excludes and differentiates from the term“compression” if the word “compression” is used to indicate theapplication of salt solutions to gel monoliths to open the pores of suchgel monoliths. Another way of solving this problem is to introducehydrophilicityto the reversed phase media to result in swelling andprevent the shrikage of the polymer in highly hydrophilic environmentduring the separation run.

More specifically, a polymerization mixture is applied to a column inthe preferred embodiment or to some other suitable mold andpolymerization is initiated within the column or mold. The column ofmold is sufficiently sealed: (1) to avoid unplanned loss by evaporationif polymerization is in an oven; or (2) to avoid contamination ordilution if polymerization is in a water bath. During polymerization,pressure is applied to the polymerization solution. Preferably thepressure is maintained at a level above atmospheric pressure to preventthe formation of voids by shrinkage until polymerization has resulted ina solid plug of separating medium or polymerization is completed. Theinner surface of the column or mold with which the polymerizationsolution is in contact during polymerization may be non-reactive or maybe treated to increase adhesion to the surface of the plug.

The polymerization mixture in some embodiments includes: (1) selectedmonomers; (2) for some types of columns, an additive; (3) an initiatoror catalyst; and (4) a porogen or porogens to form separation-effectiveopenings. In some embodiments function groups can be added before orafter polymerization. The porogen, initiator, functional group to beadded, additives, and reaction conditions and the monomer and/or polymerare selected for a specific type of column such as reverse phase, weakcation, strong cation, weak anion, strong anion columns, affinitysupport, normal phase, solid phase extraction and catalytic bed. Theselection of components of the polymerization mixture is made to providethe desired quality of column.

A chromatographic column in accordance with this invention preferablyincludes a casing having internal walls to receive a permeablemonolithic polymeric plug in which the separation-effective openings orsurface features are of a controlled size formed in the polymer by aporogen in the polymerization mixture and are controlled in size bypressure during polymerization. This plug serves as a support for asample in chromatographic columns. The permeable monolithic polymericplug has smooth walls with no visible discontinuity in the plug wall andsubstantially no discontinuity or opening within the plug. Discontinuityin this specification means a raised portion or opening or depression orother change from the normal smoothness or pattern sufficient in size tobe visible with the unaided eye. In this specification, the term“size-compensated polymers” or “size-compensated polymeric” meansmonolithic polymeric permeable material having separation-effectiveopenings in which discontinuities lack of homogeneity in theseparation-effective openings have been prevented by the methodsreferred to in this specification such as for example applying pressureduring polymerization or after polymerization during exposure to polarsolutions in the case of some types of columns or by using a column thatis prevented from further shrinkage in the presence of an aqueoussolution by the application of pressure in the presence of the aqueoussolution either during washing with an aqueous solution or during use ina separation operation using an aqueous solution.

One embodiment of column is made using a temperature controlled reactionchamber adapted to contain a polymerization mixture duringpolymerization and means for applying pressure to said polymerizationmixture in said temperature controlled reaction chamber. Thepolymerization mixture comprises at least a polymer forming material anda porogen. In one embodiment, the pressure is applied by a movablemember having a smooth surface in contact with the polymerizationmixture under external fluid or mechanical pressure, although pressurecan be applied directly to the polymerization mixture with gas such asnitrogen gas or with a liquid under pressure.

An embodiment of reversed phase media have been formed with differenthydrophobicity, and hydrophilicity from the prior art. The reversedphase media include polystyrenes, polymethacrylates and theircombinations. These media are prepared by direct polymerization ofmonomers containing desired functionalities including phenyl, C4, C8,C12, C18 and hydroxyl groups or other combination of hydrophobic andhydrophilic groups to have different selectivity and wetability inaqueous mobile phase. The polymerization conditions and porogens areinvestigated and selected to give the high resolution separation oflarge molecules, in particular, the proteins, peptides, oligonucleotidesand synthetic homopolymers. In one embodiment a reversed phase media isbased on poly(styrene-co-divinylbenzene). In another embodiment of thispatent, a reversed phase media is based on poly(stearylmethacrylate-co-divinylbenzene). In another embodiment, a reversed phasemedia is based on poly(butyl methacryalate-co-ethylene glycoldimethacrylate).

A reverse phase plug with exceptional characteristics is principallyformed of copolymers of crosslinkers including divinylbenzene (DVB), andethylene glycol dimethacrylate and monomers including styrene (ST) ormethacrylates (MA) containing different carbon chain length. Generally,the best results are when the crosslinkers are greater than 40 percentby weight Preferably the ratio of divinylbenzene and styrene is a valueof divinylbenzene in a range between 7 to 1 and 9 to 1 and preferably 4to 1 by weight, but may instead be 64 DVB or 40 percent styrene and 72percent by weight DVB or 1 g divinylbenzene, 1 g styrene. The column mayalso be in the range of ratios between 17 to 3 and 19 to 1 andpreferably 9 parts divinylbenzene to 1 part styrene. Monomers withhydrophilic functional groups can be added to reduce shrinkage of thepolymeric medium in aqueous mobile phase to prevent the wall effectduring separations. The content of DVB in total monomers is preferablyfrom 40% to 100%. In one preferred embodiment, the content of DVB is 80%(which is the highest commercially available) to improve the loadingcapacity of the column. The plug may also include methacrylates withhydrophobic surface groups or instead of being a vinyl compoundincluding urea formaldehyde or silica.

Ion exchange plugs are formed principally of methacrylate polymers. Aweak anion exchange plug is principally formed of polymers of glycidylmethacrylate (GMA) and of ethylene glycol dimethacrylate (EDMA). Astrong anion exchanger plug is principally polymers of glycidylmethacrylate, 2-(acryloyloxyethyl) trimethylammonium methyl sulfate(ATMS), ethylene glycol dimethacrylate. The polymerization mixture mayalso include 1, 4-butanediol, propanol and AIBN. A weak cation exchangerplug is formed principally of glycidyl methacrylate, acrylic acid (M)and ethylene glycol dimethacrylate. A strong cation exchanger plug isformed principally of glycidyl methacrylate,2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and ethylene glycoldimethacrylate. In all these ion exchangers, functional groups can beadded before or after the plug is formed. The content of EDMA in totalmonomers is preferably from 40% to 80%.

An increase of the content of crosslinker, such as EDMA, increases therigidity of the column by reducing the swelling of the media in aqueousphase. Each of the polymerization mixtures is modified under thepressurized polymerization to obtain high flow rate and high resolutionat both high and low flow velocity. The coupling of copolymerization ofthe monomers containing desired functional groups for interaction andthe controlled modification of other functional monomers to contain thedesired interactive functional groups increases the capacity of thecolumn while improving the rigidity of the separation media. Thiscontrolled modification may also improve the hydrophilicity of thecolumns in general by covering the potential hydrophobic surface areawith hydrophilic functional groups. The modification conditions arechosen to not only provide the higher capacity and higher hydrophilicityof the media but also to prevent the swelling of polymer matrix inaqueous solution, which happens in other highly hydrophilic polymermatrices including both beads and monolith.

The polymer plugs may be formed in a column of any size or shapeincluding conventional liquid chromatographic columns that may becircular cylinders, or coiled, bent or straight capillary tubes, ormicrochips or having any dimension or geometry. The sample or mixture tobe separated into its components is injected into the column and theliquid phase is moved through the column to separate the sample into itscomponents. The components may be detected and/or collected in afraction collector and/or inserted into another device such as a gaschromatograph or mass spectrometer. In one embodiment, a plurality ofcolumns is connected in parallel in a chromatographic system thatincludes a pumping system, solvent system and detecting system. Thecolumns are permeable polymeric columns with high reproducibility so asto enable them to work together for related separations. In oneembodiment, chromatographic discs or plugs having diameters much greaterthan 25 mm are produced.

In one embodiment, the reaction is controlled by independent means suchas for example electromagnetic radiation such as for example UV-vis,X-ray, or γ-ray instead of or in addition to reliance only on time,temperature of a water bath and the reactants in the polymerizationmixture. In one form of this embodiment, heat may be added from a heatsource or removed by cooling means in contact with a significantly largeportion of coolant of the thermal mass and in the reactor under thecontrol of feedback to maintain the temperature of the reaction mass inthe desired temperature range or to vary it during the reaction ifdesired. In another form, variable intensity or variable wavelengthX-rays may be used to control the polymerization rates of the mixingreactions at a rate such that the exotherm is under control. X-rayradiation penetrates the column to impart energy throughout the columnor at a selected location to increase of decrease polymerization rates.This may be done by irradiating the monomer sufficiently to disassociateits double bonds to make monomers free radicals and thus increase theirreactivity. Another way is to use an initiator sensitive to theradiation that is activated by the radiation in the temperature regionto be used for the reaction mass. The initiator is chosen to have anactivation time and temperature considerably less than that of themonomers alone. Because the initiator forms free radicals only uponradiation of sufficient intensity, the radiation may be used to controlthe polymerization reaction independently of the other factors. Anotherway is to use the radiation sensitizers or scintillators in combine withphoto initiators to initiate the polymerizations. The radiationsensitizers such as x-ray scintillators transfer the energy ofradiations to photo-initiators by luminescence of the photos at thedesired wavelength after absorbing the radiation energy transferredthrough the solvents. The wavelength of the luminescence should be thesame as the absorption wavelength of the photo-initiator.

Polymerization using irradiation such as x-ray is used for preparingmonolithic materials with cross sections from micrometers to meters.X-rays can penetrate the materials in depth. Both organic and inorganicpolymers can be prepared using x-ray or γ-ray. High energy x-ray andγ-ray can travel the materials in high depth. Low energy to medium x-raypenetrates the materials in less depth resulting in a longerpolymerization time but is safer to use. In one embodiment, a lowerenergy x-ray is used to initiate the polymerizations using thecombination of x-ray scintillator and photo initiators. We havediscovered that non-thermal (photoinitiation) control of polymerizationtimes from less than 12 hours to more than one week providessatisfactory chromatographic columns. Thermal polymerization of columnsusually suffers from runaway exothermic reaction and extreme temperaturegradients with columns over 20 mm in diameter. This causesununiformities which degrade chromatographic properties. The slower,controlled, polymerization rate available with x- or γ-rays, or even UVcauses a slower polymerization with tolerable rates of exotherm whilestill maintaining reasonable rates of polymerization. Thermal gradientsto exotherm maybe made small enough to not degrade the properties ofcolumns over 1 meter in diameter.

Excessive rates of exotherm and resulting process (polymerization)temperature and temperature gradient may be prevented with choice of astabilizing additive. This stabilizing additive should have propertiessuch that the reaction can proceed freely up to rate at which thedesired polymer is formed, but not at a higher rate producing too high atemperature. For example, with peroxide initiatorsDisterylhiodipropionate (DSTDP) quenches the hydroxyl radical whichresults from a side reaction, which later would go on to produce thefurther heat per event compared to the main reaction. Another approachis to use a stabilizer for the main reaction. This stabilizer isselected for limited solubility in the primary solvents or activity atthe reaction temperature and more solubility above the reactiontemperature. Under analogous conditions a stabilizer, preferentiallysoluble in the porogen and having a temperature dependent of solubilityor activity may be used.

It is desirable to scale up the size of the column to have higher volumeof media is highly desired in preparative chromatography and catalyticreactors. In one embodiment of the invention, the large diameter columnis prepared by two staged polymerization inside the column. First,multiple thin cylindrical columns with the diameter smaller than that ofthe targeted column are prepared in a mold under pressure or withoutpressure. The thin columns are placed inside a large column filled withthe same polymerization solution as used in formation of the thincolumns. The thickness in one side of the thin column should not exceedthe 8 mm which is the known maximum to prevent the formation oftemperature gradient due to the difficulty in heat dissipation duringexothermic polymerization. The temperature gradient results in varyinhomogeneous pore size distribution which is detrimental tochromatography use.

In making size-compensated polymers for use in separation systems, thecharacteristics for a given type of separation can be tailored with agiven polymer to the application, by altering the amount of pressureapplied during polymerization or and in the case of some polymers suchas used in forming reverse phase separation media applying pressure whenused or when otherwise brought into contact with a polar solvent such asan aqueous solvent or washing fluid. After the nature of the polymeritself has been selected for a class of applications, columns can bemade and tested. Based on the tests, the characteristics can be alteredin some columns by applying pressure. It is believed that theapplication of pressure in some columns increases the uniformity ofparticle size and either because of the change in particle size of forother reasons, the size distribution and uniformity of separationeffective openings throughout the polymer is increased. The increase inhomogeniety of the particle size and pore size improves resolution. Anincrease in pressure generally improves capacity and resolution and thepressure-time gradient. It is believed that in some columns microporesare greatly reduced or eliminated thus reducing zone spreading by theapplication of pressure during polymerization and/or during use orwashing of the polymer with polar solutions.

From the above description it can be understood that the novelmonolithic solid support of this invention has several advantages, suchas for example: (1) it provides chromatograms in a manner superior tothe prior art; (2) it can be made simply and inexpensively; (3) itprovides higher flow rates for some separations than the prior artseparations, thus reducing the time of some separations; (4) it provideshigh resolution separations for some separation processes at lowerpressures than some prior art processes; (5) it provides high resolutionwith disposable columns by reducing the cost of the columns; (6) itpermits column of many different shapes to be easily prepared, such asfor example annular columns for annular chromatography and prepared inany dimensions especially small dimensions such as for microchips andcapillaries and for mass spectroscopy injectors using monolithicpermeable polymeric tips; (7) it separates both small and largemolecules rapidly; (8) it can provide a superior separating medium formany processes including among others extraction, chromatography,electrophoresis, supercritical fluid chromatography and solid supportfor catalysis, TLC and integrated CEC separations or chemical reaction;(9) it can provide better characteristics to certain known permeablemonolithic separating media; (10) it provides a novel approach for thepreparation of large diameter columns with homogeneousseparation-effective opening size distribution; (11) it provides aseparation media with no wall effect in highly aqueous mobile phase andwith improved column efficiency: (11) it improves separation effectivefactors; and (12) it reduces the problems of swelling and shrinking inreverse phase columns.

BRIEF DESCRIPTION OF THE DRAWINGS

The above noted and other features of the invention will be betterunderstood from the following detailed description, when considered withreference to the accompanying drawings in which:

FIG. 1 is a schematic diagram of one embodiment of a process for makinga chromatographic column in accordance with an embodiment of theinvention;

FIG. 2 is an assembly of a fixture for applying pressure to a glasscolumn during polymerization;

FIG. 3 is an assembly of another fixture for applying pressure to astainless steel column during polymerization;

FIG. 4 is an assembly of still another fixture for applying pressure toa glass column during polymerization;

FIG. 5 is a Scanning Electron Microscopy (SEM) picture of the strongcation exchanger polymerized inside a cylinder column under 120 psihydraulic pressure.

FIG. 6 is a chromatogram showing peaks from a protein sample with acolumn in which distortions have been avoided by pressure duringpolymerization;

FIG. 7 is a photograph showing three columns with the one on the leftmade with pressure during polymerization and the two on the rightpolymerized without pressure;

FIG. 8 is a block diagram of a chromatographic system with an array ofcolumns and having reproducible characteristics which are similar toeach other in the array.

FIG. 9 is a chromatogram showing the chromatography separation achievedwith medium energy (110 kEV)x-ray irradiated polymerization.

FIG. 10 is a top view of a UV or visible light polymerization apparatusfor chromatographic columns;

FIG. 11 is a sectional side elevational view of the apparatus of FIG.10.

FIG. 12 is a schematic elevational view of an x-ray polymerizationapparatus for chromatographic columns;

FIG. 13 is a top view of a portion of the apparatus of FIG. 12;

FIG. 14 is an elevational sectional view of a portion of the apparatusof FIG. 12 taken through lines 14-14; and

FIG. 15 is an elevational sectional view taken through lines 15-15 ofFIG. 13.

DETAILED DESCRIPTION

Broadly, a polymerizable mixture is placed in a container with a porogenor solvent and polymerized to form a plug having separation-effectiveopenings for use in a separation system such as for example achromatographic column. Advantageously, the polymerization is done in acontainer in which the plug is to be used such as a chromatographiccolumn or extraction chamber or the like. In one embodiment of theinvention, the mixture is polymerized while compensating for the effectof shrinkage during poly merization to form a size compensated polymericplug. In other embodiments shrinkage is avoided by applying pressure tomaterials that tend to swell in the presence of water to form anotherembodiment of size compensated polymeric plug. In still otherembodiments separation-effective opening size and distribution iscontrolled by pressure and/or by selection of the ingredients of thepolymerization mixture and/or by processes using external influencessuch as electromagnetic radiation. Shrinking is compensated for oravoided because it may causes enlarged voids adjacent to the wall of thecontainer and have a deleterious effect on pore size distribution withinthe column.

In a preferred embodiment, the compensation is accomplished by applyingpressure during polymerization to at least maintain the integrity of thematerial having separation-effective openings as it shrinks duringpolymerization. Maintaining the column at atmospheric pressure toaccommodate shrinkage may not prevent the formation of voids in everycase and may provide poor reproducibility. The polymers, monomers,initiators and porogens have been selected to improve thecharacteristics of the column and may be used with the embodiments ofpolymerization under pressure during polymerization or with otherprocesses. In another embodiment, pressure is applied to the plug afterit has been formed and after or during swelling caused by otherreactions such as washing with an aqueous solution after polymerizationin the case of some reverse phase plugs. The surface of the column ormold with which the polymerization solution is in contact duringpolymerization may be non-reactive or may be treated to increaseadhesion.

In one embodiment, the polymerization mixture includes at least onevinyl compound and a porogen. An initiator is included in thepolymerization mixture or an initiation process is applied to a vinylmonomer and a porogen to form a monolithic plug for a chromatographiccolumn or other device using a polymeric plug for separation. Generallythe polymerization mixture includes, in addition to the vinyl monomer, avinyl polymer, or mixture of monomers and polymers, an initiator and aporogen. However, other approaches to polymerization withoutincorporating an initiator in the polymerization mixture are known inthe art and can be used, such as radiation to form polymers. Moreover,some of the aspects of this invention may be applied to monomers andpolymers in a polymerization reaction other than vinyl groups such asfor example urea formaldehyde or silica to form urea formaldehyde orsilica plugs.

A chromatographic column formed by these processes includes a supporthaving internal walls to receive a permeable monolithic polymeric plughaving separation-effective openings of a preselected size distributionformed during polymerization and controlled in size partly by pressureduring polymerization and partly by selection of the components andproportions of the components of the polymerization mixture. Forexample, separation-effective opening size is controlled by the amountand type of porogen in the polymerization mixture in proportion to theamount of some of the other ingredients. The pressures may be selectedin a range of slightly above atmospheric pressure to a value within thestrength of the walls of the column. The permeable monolithic polymericplug has smooth walls and substantially no micropores within the plug.The plugs may have surface functional groups. For example, hydrophobicsurface groups such as phenolic groups may be added to decrease swellingwith aqueous based solvents in reverse phase plugs but capacity may alsobe decreased in this case. Similarly, hydrophillic surface groups may beadded to increase capacity in reverse phase plugs.

One embodiment of permeable, monolithic, polymeric plug that is free ofmicropores or channeling openings in the walls over is formedprincipally of vinyl polymers although many other polymers may be usedin practicing the invention. A weak ion exchange permeable monolithicpolymeric plug is principally formed of polymers of methacrylate such asglycidyl methacrylate and ethylene dimethacrylate in the ratios byweight of a value in the range between 2.5 and 3.5 to a value between1.8 and 2.2 and preferably 3 to 2. A reverse phase permeable monolithicpolymeric plug with exceptional characteristics is principally formed ofpolymers of divinylbenzene, and of styrene. Preferably the ratio ofdivinylbenzene and styrene is approximately in a range of a valuebetween 3.5 and 4.5 to a value between 0.8 and 1.2 and preferably 4 to 1by weight, but may instead be 64 DVB (divinylbenzene) or 40 percentstyrene and 72 percent by weight DVB or in the ratio of divinylbenzeneto styrene in the range of a ratio of 2 to 3 and a ratio of 3 to 2 orpreferably 1 to 1. The column may also be in the ratio of divinylbenzeneto styrene in a range of the ratio of 8 to 1 and 10 to 1 and preferably9 to 1. One hundred percent DVB is also preferred.

In FIG. 1 there is shown a block diagram of one embodiment 10 of amethod for making chromatographic columns comprising the step 12 ofpreparing a polymerization mixture, the step 14 of polymerizing themixture, the step 16 of preparing the column for chromatographic run andthe step 18 of performing a chromatographic run. The polymerizationmixture used in step 14 of polymerizing a mixture includes a monomer andor polymer capable of polymerization, an initiator or initiation processsuch as radiation, and a porogen, many of which are known in the art.The step 12 of preparing the chromatographic mixture, the step 14 ofpolymerizing, the step 16 of preparing for a chromatographic run and thestep 18 of performing the chromatographic run all may take differentforms. Some of these variations will be described hereinafter.

In the step 14 includes the substeps 20 of reacting the polymerizationcompound while compressing it to reduce voids, the substep 22 of washingthe polymer, and in some embodiments, the step 24 of reacting thepolymer to add certain functional groups. While a workable column may beobtained without compressing the polymerization mixture whilepolymerizing, significant improvements have been obtained by applyingthis compression. These improvements have been significant enough so asto make the difference between a competitive commercial column and onewhich would not be competitive in some types of columns and for someapplications The voids and inhomogeneous separation-effective openingsthat are prevented from forming by this compression may result from theinhomogeneous distribution of the empty space created by shrinkage ofthe polymer during polymerization in a sealed container. Theinhomogeneous distribution of the empty space may be due to thedifferences in surface tension between the column wall surface, polymersurface, nitrogen and porogens. The compression must be sufficient totake up this shrinkage and thus reduce the total volume of the columnduring polymerization. This process may also affect theseparation-effective opening size in the column and can be used in astep by step process to create variations in separation-effectiveopening size if desired.

The washing step 22 is a conventional step intended to remove porogensand unreacted monomers or other ingredients that may be used for aspecific column but are not intended to remain in the column. This stepmay be followed by reacting in a manner to add functional groups such asthe groups described above that are important if the column is intendedto separate proteins. In some embodiments, the washing step causesswelling of the plug followed by later shrinkage. Because the shrinkagemay cause channeling voids, pressure is applied to the swollen plug inone embodiment to prevent the formation of voids during shrinking.

In FIG. 2, there is shown a block diagram of a polymerizing apparatus 28having a pressure source 23, a pressure transfer mechanism 25 forapplying pressure to a polymerization mixture 32 compression piston 27and a confinement vessel 21. In the preferred embodiment, the source ofpressure 23 is a regulated source of constant hydraulic pressure butother sources such as a spring or source of air or of an inert gas maybe used. Similarly, in the preferred embodiment, the mechanism 25 is apiston with a smooth surface to provide a smooth surface to thepolymerized plug that the piston surface has pressed against duringpolymerization but other sources such as a gas applied directly to thepermeable monolithic polymeric plug may be used. In the preferredembodiment, the compression piston 27 moves inwardly into the columndepartment 21 to exert pressure on the polymerization mixture within thecompartment 32 during the polymerization reaction. When thepolymerization reaction is complete, the porogen can be removed by asolvent pumped through the column. The polymerization occurs in atemperature controlled environment 29, which is the preferred embodimentis a water bath but can be any such temperature control mechanism suchas a heated chamber. The materials for this device can be anyconventional materials know in the art.

In FIG. 3 there is shown a sectional view of one embodiment of thepolymerizing apparatus 28 having a metal column casing 1022, aconfinement vessel 88, a transfer mechanism 25, a compression piston 112and a pressure cap 80. The metal column 1022 is tightly held against theconfinement vessel 88 with a seal 1021 between them. Compression of theseal 1021 is provided by a shoulder 1052 in the barrel 122 and wrenchflats 1023 of the apparatus, which is attached to the column 1022 withthreads 1053, thus providing a leak free connection between the column1022 and the confinement vessel 88. A transfer mechanism 25 consistingof a compression piston 112, an o-ring 110, a rod 106, a retainingcollar 104, another o-ring 100, and a hydraulic piston head 602, all ofwhich are arranged and fitted into the barrel 122 such that thecompression piston 112 and o-ring 110 form a tight seal inside theconfinement vessel 88. The pressure cap 80 contains a fluid inlet port33 fitted to the barrel 122, with a gasket 94 between them.

The pressure cap 80 and the barrel 122 are tightly connected, preventingthe leakage of pressurized fluid applied through the fluid inlet 33. Thetransfer mechanism 25 is then positioned as shown, creating a volume inthe confinement vessel 88. The square of the ratios of the insidediameter of the barrel 122 at o-ring 100 to inside diameter ofconfinement vessel 88 provides a pressure multiplication factor. Theopposite end of the column 1022 is filled with the polymerizationreactants in the column compartment 32, and a containment plug 1024 isfitted in the opening. A containment cap 604 is threaded onto the column1022, forcing the containment plug to seal the opening. Although thepreferred embodiment here shows a tight fitting plug 1024 to provide thesealing, an alternate sealing arrangement, such as an,o-ring, could aseasily be used to provide either a face seal or a radial seal. The fluidinlet 33 is connected to a controlled pressure source, such as acontrollable fluid pump or regulated bottle of compressed gas.

This description of the preferred embodiment employs a fluid source;either compressed gas or compressed liquid applied through the fluidinlet 33, however the compressive force could as easily be supplied byalternate means; such as, but not limited to a spring pressing on thetransfer mechanism 25, weights stacked on the transfer mechanism 25utilizing gravity to provide the compression, or centripetal forcearranged to cause the transfer mechanism 25 to compress the monolithicpolymeric column material inside the column compartment 32.

Once assembled, the apparatus 28 is placed in a temperature-controlledenvironment 27, which is a thermally controlled water bath in thepreferred embodiment. Fluid pressure is then applied through the fluidinlet port 33, which is contained by the pressure cap 80, the gasket 94the hydraulic piston head 602 and the o-ring 100. This applied forcecauses the hydraulic piston head 602 to move away from the pressure cap80, and exerts force on the end of the rod 106. This rod 106communicates the force to the compression piston, applying compressivepressure to the monolithic polymeric column material, preferably at thesmooth surface 1101. This smooth surface causes a continuous, uniformsurface to be created on the monolithic polymeric material exposed tothe analytical fluids in the ultimate application and reduces theadhesion of the monolithic polymeric column material to the compressionpiston 112.

The containment plug 1024, column 1022, seal 1021 confinement vessel 88,and the compression piston 112 confine the monolithic polymericmaterial. As the chemical reaction proceeds, the volume of themonolithic polymeric material decreases, and the transfer mechanism 25moves further into the confinement vessel 88. Air trapped between theo-ring 100 and the o-ring 110 is allowed to escape through an air escapeopening 603 in the barrel 122. The compression of the reactant materialsin this manner prevents the formation of undesirable voids in themonolithic polymeric material and eliminates wall effects between themonolithic polymeric material,and the column 1022, which would reducethe performance of the column in use.

As the reaction proceeds and the monolithic polymeric material volumereduces, the compression piston moves closer to the column 1022. Nearthe end of the polymerization, the retaining collar 104 contacts theshoulder 608 in the barrel 122, halting the forward motion of thetransfer mechanism 25. Crushing of the newly formed monolithic polymericmaterial is prevented by this action. At this position, the smoothsurface 1101 of the compression piston 112 is approximately even withthe end of the column, and the monolithic polymeric material fills thecolumn 1022 without undesired voids in the material or wall effectsbetween the material and the column 1022. Using the wrench flats 1023and 604, the polymer apparatus 28 is separated from the column 1022 asan assembly. Chromatographic fittings are then installed on both ends.

In FIG. 4 there is shown a sectional view of another embodiment ofpolymerizing apparatus 28A similar to the embodiment of polymerizingapparatus 28 having a glass column casing 922, a piston head assembly401, a displacement piston 40 and a containment plug 923. In someanalytical chemistry applications, the wetted surfaces must not containmetal components. Although the present discussion of this preferredembodiment refers to a glass column 922 and plastic pieces, anynon-metallic material; such as, but not limited to glass, ceramic, orplastic which provides acceptable mechanical properties can be used.Further, the discussion here refers to the pressure applied beingprovided by a suitable fluid pressure source, alternative means ofproviding compression; including, but not limited to springs, weights,or mechanical means could as easily be used.

The piston head assembly 401 comprises a piston 76, an o-ring 38 and anintermediate portion 50, assembled and filted into the column 922. Aplunger assembly 20 consisting of the displacement piston 40 and ano-ring 64 are assembled and fitted into the hydraulic cylinder portion21 such that the recess 92 is away from the fluid inlet port 33 in thehydraulic cylinder portion 21. This plunger assembly 30 is pushed fullyinto the displacement chamber 60.

The hydraulic cylinder portion 21 and plunger assembly 30 are thenthreaded onto the column 922. Using a suitable tool, the piston assembly401 is pushed into the hydraulic cylinder portion 21 until the annularshoulder 42 contacts the displacement piston 40, with the reduceddiameter neck 48 fitting into the recess 42. The column 922 is filledwith the reactant, and the containment plug 923 is inserted into theopen end of the column 922. A containment cap 924 is then threaded ontothe end of the column 922, tightly holding the containment plug 923 tothe column 922. Although this embodiment utilizes a tight fit betweenthe containment plug 923 and the column 922, alternate methods;including, but not limited to an o-ring creating a face or radial sealcan as easily be used.

A fluid pressure source is then applied through the fluid inlet port 33.The fluid is contained within the displacement chamber 60 by thehydraulic cylinder portion 21, the displacement piston 40 and the o-ring64. The assembled components are then placed in a temperature-controlledenvironment 27. For this embodiment, a thermally controlled water bathwas used, but any suitable method of controlling the reactiontemperature can be employed.

The application of such fluid pressure causes the displacement piston 40to move away from the fluid inlet port 33. This movement of thedisplacement piston 40 applies force to the annular shoulder 42 of theintermediate portion 50, which then applies pressure to the monolithicpolymeric material in the column 922 through the piston 76. As thereactant chemicals polymerize, the volume decreases. With the controlledapplication of pressure to the monolithic polymeric material preventsthe formation of undesirable voids within the monolithic polymericmaterial and the formation of wall effects between the monolithicpolymeric material and the wall of the column 922. As the reactionprogresses, the piston 76 moves further into the column 922 to displacethis reduction in volume. A smooth surface 74 on the piston creates auniform surface of the monolithic polymeric material to provide aconsistent interface to the analytic fluids in its final use, and toprevent the monolithic polymeric material from adhering to the surfaceof the piston 76.

Near the end of the reaction, the annular shoulder 42 comes in contactwith the end of the column 922, preventing any further movement of theintermediate portion 50 into the column 922. This prevents the crushingof the monolithic polymeric material after the voids and wall effectshave been eliminated. This annular shoulder 42 also limits the distancethat the piston can travel, allowing control the porosity and size ofthe resultant monolithic polymeric material in the column 922.

After the polymerization is completed, the hydraulic cylinder portion 21is removed from the column 922, together with the displacement piston 40and its o-ring 64. The confinement cap 924 and confinement plug 923 arethen removed, and finally the piston head assembly 401 is removed.Chromatographic fittings are then installed on both ends.

It is also possible to provide compression on the reactant chemicals bythe direct application of compressed gas directly to the reactantchemical's surface. Such a method is considered inferior to the abovetechniques because the surface of the resultant monolithic polymericmaterial will not be smooth or even, and may be more porous than thebody of the monolithic polymeric material, when particular columnformats are chosen. In other column formats the direct application ofgas may be more applicable. It may be necessary to cut off the end ofthe polymer rod to achieve high resolution separation.

In FIG. 5 there is shown a Scanning Electron Microscopy (SEM) picture ofa strong cation exchanger polymerized inside a column casing under 120psi hydraulic pressure magnified 9,000 times to show globules ofparticles with no pores but with channels between them having highsurface area because of the irregular surface area and to a lesserextent the more stacked-plate like configurations of the globules ofparticles. The rough surface area of the particles with projectionscovering their surface area shows signs that may indicate growth byaccretion.

In FIG. 6, there is shown a chromatogram having peaks from a proteinsample separated in a column in which the problems of swelling andshrinking avoided by the application of pressurel. The peaks aredistinctive and relatively high with good resolution. This particularchromatogram is for gradient elusion at a flow rate of 3 ml/min on aprotein sample of conalbumin, ovalbumin and tripsin inhibitor using a0.01 MTris buffer of pH 7.6 as one solvent and a 1 M sodium chloride asthe other solvent with a gradient of 0 to 50 percent the second solventin 5 minutes time. The back pressure is 250 pounds per square inch inthis column whereas a column without such compensation would be expectedto have a higher back pressure for the same gradient.

In FIG. 7, there is shown three plugs with the one on the left made withpressure during polymerization and the two on the right polymerizedwithout pressure. FIG. 7 illustrates the discontinuities formed on thesurface of columns caused by shrinkage during formation of the column.There are similar discontinuities inside the column in the form ofrelatively large openings unpredictably spaced. These figures alsoillustrate that the discontinuities can be removed, resulting in betterreproducibility between columns of the same composition and the samesize and improved resolution during chromatographic runs.

In FIG. 8, there is shown a block diagram of a preparatory liquidchromatographic system 101 having a pumping system 121, a column anddetector array 141, a collector system 117, a controller 119, and apurge system 123. The column and detector array 141 includes a pluralityof columns with permeable plugs in them. Preferably the plugs aresize-compensated polymeric plugs. The pumping system 121 suppliessolvent to the column and detector array 141 under the control of thecontroller 119. The purge system 123 communicates with a pump array 135to purge the pumps and the lines between the pumps and the columnsbetween chromatographic runs. The pump array 135 supplies solvent to thecolumn and detector array 141 from which effluent flows into thecollector system 117 under the control of the controller 119. Thecontroller 119 receives signals from detectors in the column anddetector array 141 indicating bands of solute and activates the fractioncollector system 117 accordingly in a manner known in the art. Onesuitable fraction collector system is the FOXY7 200 fraction collectoravailable from Isco, Inc., 4700 Superior Street, Lincoln, Nebr. 68504.

To supply solvent to the pump array 135, the pumping system 121 includesa plurality of solvent reservoirs and manifolds, a first and second ofwhich are indicated at 131 and 133 respectively, a pump array 135 and amotor 137 which is driven under the control of the controller 119 tooperate the array of pumps 135. The controller 119 also controls thevalves in the pump array 135 to control the flow of solvent and theformation of gradients as the motor actuates the pistons of thereciprocating pumps in the pump array 135 simultaneously to pump solventfrom a plurality of pumps in the array and to draw solvent from thesolvent reservoirs and manifolds such as 131 and 133.

While in the preferred embodiment, an array of reciprocating pistonpumps are used, any type of pump is suitable whether reciprocating ornot and whether piston type or not. A large number of different pumpsand pumping principles are known in the art and to persons of ordinaryskill in the art and any such known pump or pumping principle may beadaptable to the invention disclosed herein with routine engineering inmost cases. While two solvents are disclosed in the embodiment of FIG.1, only one solvent may be used or more than two solvents.

To process the effluent, the collector system 117 includes a fractioncollector 141 to collect solute, a manifold 143 and a waste depository145 to handle waste from the manifold 143. One or more fractioncollectors communicate with a column and detector array 143 to receivethe solute from the columns, either with a manifold or not. A manifoldmay be used to combine solute from more than one column and deposit themtogether in a single receptacle or each column may deposit solute in itsown receptacle or some of the columns each may deposit solute in its owncorresponding receptacle and others may combine solute in the samereceptacles. The manifold 143 communicates with the column and detectorarray 141 to channel effluent from each column and deposit it in thewaste depository 145.

Because the system of FIG. 8 includes an array of columns each involvedin a similar task, reproducibility of the column is particularlyimportant since it is desirable for each column performing a single taskto have characteristics as similar to all of the other columnsperforming that task as possible. Consequently, there is a substantialadvantage in any group of columns that are intended to cooperate in theperforming of a separation of samples closely related to each other, thepermeable polymeric columns of this invention have particularapplication.

To make a column or other device having a polymeric plug as a separatingmedium without openings in the side walls or large openings in the bodyof the plug, one embodiment of polymerization equipment includes atemperature controlled reaction chamber adapted to contain apolymerization mixture during polymerization and means for applyingpressure to said polymerization mixture in said temperature controlledreaction chamber. The polymerization mixture comprises a monomer,polymer and a porogen. In a preferred embodiment the means for applyingpressure is a means for applying pressure with a movable member. Thepolymerization mixture comprises includes a cross-linking reagent and across-linking monomer. The rigidity, capacity and separation-effectiveopening distribution are controlled by the amount of cross-linkingreagent, monomer, and pressure.

The polymerization takes place in a closed container to avoid loss ofsolvent in the case or an oven or to avoid dilution or contamination ofthe mixture with water in the case of a water bath reaction chamber.Pressure is applied during polymerization of some mixtures such asmixtures for ion exchange columns to balance vacuum formed by shrinkage.The polymer plug is washed after polymerization to remove the porogen.In the case of some polymer, the plug may have a tendency to swellduring washing or during a chromatographic run if aqueous solutions areapplied such as if the plug is a reverse phase plug.

To separate a mixture into its components, a permeable polymeric plug isformed as described above. It may be formed in a column of any size orshape including conventional liquid chromatographic columns that areright regular tubular cylinders, or capillary tubes, or microchips orhaving any dimension or geometry. A sample is located in juxtapositionwith the plug and the components of the sample are separated one fromthe other as they are moved through the plug.

For example, to separate proteins from a mixture of proteins in a sampleby liquid chromatography, a column is formed as a plug and polymerizedin place with a porogen. Shrinkage is compensated for before use. Thesample is injected into the column and a solvent caused to flow throughthe column, whereby the sample is separated into its components as it iscarried through the plug. In one embodiment, a plurality of samples areseparated simultaneously in separate columns with high reproducibility.

One embodiment of chromatographic columns used in separation processeshave a chromatographic casing with internal casing walls and have apermeable monolithic polymeric plug in the casing walls. The plug is apolymer having separation-effective openings which may be of acontrolled size formed in the polymer by a porogen in the polymerizationmixture before polymerization and controlled in size at least partly bypressure during polymerization. The permeable monolithic polymeric plughas smooth walls and substantially no pores within the permeablemonolithic polymeric plug. In the preferred embodiment, the plug isformed of vinyl polymers but may be formed of others such as ureaformaldehyde or silica. The may include surface groups such ashydrophobic groups to reduce swelling with aqueous solvents orhydrophillic groups to increase capacity.

Some examples of the proportions of the ingredients in polymerizationmixtures and the ingredients in different plugs are illustrative. Forexample, a weak ion exchanger permeable monolithic polymeric plug thatis free of channeling openings is formed principally of methacrylatepolymer. Advantageously, his permeable monolithic polymeric plug isprincipally formed of polymers of glycidyl methacrylate and of ethylenedimethacrylate in the ratio by weight in a range of ratios between 1 to1 and 7 to 3 and preferably 3 to 2.

A strong anion exchanger includes as its principal ingredients glycidylmethacrylate (GMA) and ethylene divinyl methacrylate (EDMA) in the ratioof a value in the range of 0.8 to 1.2 to a value in the range of 2.8 to3.2 and preferably a ratio of 1 to 3. The polymerization solution in thepreferred embodiment includes 0.4 grams GMA, 0.5 grams of2-(acryloyloxyethyl) trimethylammonium methyl sulfate, 1.2 grams EDMA,1.5 grams of 1,4-butanediol, 1.35 grams propanol, 0.15 grams water and0.02 grams AIBN.

A weak cation exchanger included a polymerization solution of methylmethacrylate (MMA), GMA and EDMA in the ratio of a value of MMA in therange of 4.5 to 5.5 to a value of GMA in the range of 0.8 to 1.2 to avalue of EDMA in a range of 11 to 13, and preferably a ratio of 5 to 1to 12. The polymerization solution includes 0.2 grams M, 0.5 gramsmethyl methacrylate (MMA), 0.1 grams GMA, 1.2 grams EDMA, 2.55 gramsdodecanol, 0.45 grams cyclohexanol and 0.02 grams AIBN. Afterpolymerization, this column is hydrolyzed in a 0.25 M sodiumchloroacetate in 5 M (molar) sodium hydroxide NaOH at 60° C. for sixhours.

Other examples are: (1) glycidyl methacrylate and of ethylenedimethacrylate in a ratio by weight in the range of from 1 to 1 and 2 to1; or (2) divinylbenzene and styrene in a ratio in the range of 3 to 1and 9 to 1; or (3) divinylbenzene and styrene in a ratio in the range of4 to 1 or with the amount divinylbenzene being in the range of 35percent and 80 percent by weight divinylbenzene and preferably 64percent by weight divinylbenzene; or (4) divinylbenzene, styrene andporogen are in the ratio of 8 to 2 to 15 respectively; or (5)divinylbenzene, styrene and dodecanol in a proportion within the rangeof 7 to 9 units of divinylbenzene to 1.5 to 2.5 units of styrene to13-17 units of dodecanol combined with an initiator, or (6)divinylbenzene, styrene and dodecanol in the range of 8 to 2 to 15respectively combined with an initiator; or (7) divinylbenzene, styrenedodecanol and toluene in the proportions of 7-9 to 1.5-2.5 to 9-13 to2.5-3.5 respectively, combined with an initiator; or (8) divinylbenzene,styrene dodecanol and toluene combined in the proportions of of 8 to 2to 11 to 3 respectively, combined with an initiator; or (8) glycidylmethacrylate, ethylene dimethacrylate, cyclohexanol and dodecanol in theproportions of 0.5-0.7 to 0.3-0.5 to 1-2 to 0.1-2.5, combined with aninitiator; or (9) glycidyl methacrylate, ethylene dimethacrylate,cyclohexanol and dodecanol in the proportions of 0.6 to 0.4 to 1.325 to0.175 respectively.

In general, a process of preparing a monolithic polymer support havingseparation-effective openings for a targeted application may include thefollowing steps: (1) preparing a polymerization mixture with a selectedformula; (2) placing the mixture in a container, sometimes referred toas a column in the embodiments of this inventions, with desired shapeand size; (3) sealing the column with pressurizing filtings ornon-pressure sealing; (4) polymerizing the polymerization mixture in aheating bath or oven with controlled temperature under selected pressureor without pressure; (5) taking the columns from the heating bath oroven and applying selected or specially designed fittings for thedesired function; (6) washing the porogens and soluble materials out ofthe columns with selected solvent preferably by programmed flow; (7) insome embodiments, pumping a formulated modification solutions to obtainthe desired functionality for interaction; (8) performing specialmodification in a heating bath or oven under controlled conditions; (9)washing the modification solutions out of the columns preferably with aprogrammed flow; (10) stabilizing, assembling and conditioning thecolumn for its use at desired conditions with high resolution; (11)characterizing the columns with sample separation in the targetapplication; (12) replacing the liquid in the column with selectedstorage solution. Steps 7 to 9 areas optional or repetitive depending onthe functionality of the media to be used. Steps 1 to 5 are modified andrepeated in the two or multiple staged polymerization process.

In some of the embodiments of this invention, a polymerization mixtureincludes single or plurality of: (1) monomers; (2) porogens; (3)initiators or catalysts; and/or (4) additives or fillers (optional). Thepolymerization mixture may be degassed with helium for more than 15minutes, or by vacuum, or by combination of both prior to be filled orinjected to the column. The goal of this degassing is to get rid of theoxygen inside the mixture. The oxygen can act as an inhibitor orinitiator at different situation resulting in some unpredictablebehavior of the polymerization, which is detrimental to the resolutionand reproducibility of the columns.

The suitable monomers for the above process comprise mono, di andmultiple functional monomers known in the art, preferably monomerscontaining the vinyl or hydroxyl silica functional groups, which mightbe generated in situ as an intermediate. The typical monovinyl monomersinclude styrene and its derivatives containing hydroxyl, halogen, amino,sulfonic acid, carboxylic acid, nitro groups and different alkyl chainsuch as c4, c8, c12 and c18, or their protected format which could beused to generate those functionalities before or after polymerization;and include acrylates, methacrylates, acrylamides, methacrylamides,vinylpyrolidones, vinylacetates, acrylic acids, methacrylic acids, vinylsulfonic acids, and the derivatives or these groups which could be usedto generate these compounds in situ. The mixture of these monomers canbe used. Siloxanes with hydroxyl group, vinyl groups, alkyl groups ortheir derivative and mixture thereof are preferred. The amount of themonofunctional monomers are varied from 2% to 60% of the total monomersin the embodiments of this invention. They vary dramatically depend onthe type of media.

The typical di or multifunctional monomers are preferably the di ormultiple vinyl-containing monomers with a bridging moietysuch asbenzene, naphthalene, pyridine, alkyl ethylene glycol or its oligoes.Examples of these polyvinyl compounds are divinylbenzene,divinylnaphthalene, alkylene diacrylates, dimethacrylates, diacrylamidesand dimethacrylamide, divinylpiridine, ethylene glycol dimethacrylatesand diacrylates, polyethylene glycol dimethacrylates and acrylates,pentaerythritol di-, tri-, or tetramethacrylate and acrylate,trimethylopropane trimethacrylate and acrylate, and the mixture of thesecompounds. Siloxanes with di, tri and tetrahydroxyl groups, which areoften generated in situ are also preferred in this invention. Thetypical amount of the multifunctional monomers are from 40% to 80% inthe embodiments of this invention.

The initiators comprise all the initiators known in the art such as azocompounds, peroxides. Example of the typical intiators areazobisisobutylonitrile, benzoyl peroxide,2,2′-azobis(isobutyramide)dehydrate,2,2′-azobis(2-amidinopropane)dihydrochloride. The typical amount of theinitiator is from 0.5% to 2% of the total monomers in the embodiments ofthis invention. When siloxane is used, a catalyst such as an acid isused instead of an initiator. The amount of catalyst is from milimolesto moles per liter of polymerization mixture. Other approaches topolymerization without incorporating an initiator in the polymerizationmixture are known in the art and can be used, such as radiation to formpolymers.

The porogen is any material or compound which can be removed afterpolymerization to generate separation-effective opening structures. Thetypical porogens may be used are organic solvents, water, oligomers,polymers, decomposable or soluble polymers. Some example of the organicsolvents are alcohols, esters, ethers, aliphatic and aromatichydrocarbons, ketones, di, tri, tetraethylene glycols, butane diols,glycerols or the combination of these solvents. The choice of porogensdepends on the separation-effective opening size andseparation-effective opening distribution needed.

In some embodiments, a single or a combination of porogenic solventswhich are mixable with the monomers and initiators to form a homogeneoussolution but have poor solvating power to the polymers formed is chosen.The polymerization usually starts from the initiator. The formation ofoligomers is followed by crosslinking forming crosslinked polymer ornuclei, and the continuous growth of the polymer or nuclei. Thesepolymer chains and nuclei precipitate out of the solution at the sizeallowed by the solvating power of the porogenic solvents. These polymerchains and nucleis are suspended in the solution first and form smallparticle through collision and crosslinking. The small particles areswelled by the porogens and monomers, and continue to grow by bothpolymerization and aggregation with other nucleis or particles. Thelarger particles aggregate together by collision and held in place bycrosslinking. The time and speed of the precipitation of the polymer andnuclei dramatically affect the size of particles, aggregates or clustersand the separation-effective opening size formed among these particlesand aggregates as well as the separation-effective opening sizedistribution.

It has been discovered that the combination of a very poor solvent and afairly good solvent are usually better to tune the solubility orswellability of the polymer in the solution, which result in desiredporosity and separation-effective opening size distribution. The choiceof poor solvent is more important since generation of the largeseparation-effective opening is the most important. After the generationof large separation-effective opening, it is always easier to find agood or fairly good solvent to tune the separation-effective openingsize down. It has been discovered that the alcohols or the neutralcompounds containing a hydroxyl group or multiple hydroxyl groups arethe better choice of the poor solvents for the media made ofpolystyrenes, polymethacrylates, polyacrylates, polyacrylamides andpolymethacrylamides. The solubility or solvation power can be easilytuned to using alcohols of different chain length and number of hydroxylgroups. A good solvent for the polymers can be chosen from manyconventional good solvents such as toluene, tetrahydrofuran,acetonitrile, formamide, acetamide, DMSO. They typical amount of theporogens vary from 20% to 80%, more preferably 40% to 60% in theembodiments of this invention.

The additives or fillers used in this invention are those materialswhich can add a specifically desired feature to the media. One importantcharacteristic of polymers having separation-effective openings is therigidity of the polymer. Insoluble rigid polymer particles, silicaparticles, or other inorganic particles can be added into thepolymerization mixture to strengthen the polymer havingseparation-effective openings after the polymerization. Polymers with avery large number or amount of separation-effective openings usually donot have good strength or toughness. They are fragile most of the time.The rigid particles can act as framework for the polymers. In anotherembodiment, the resolution of the large columns and reduce the problemof heat transfer during the preparation of large diameter columns arereduced by adding very mono-dispersed nonporous particles to thepolymerization mixture for. Quite often, a large diameter column isrequired for high flow preparative chromatography or catalytic bed toallow high flow rate with only low back pressure.

In an embodiment of polymer having separation-effective openings,mono-dispersed large non-porous particles or beads are packed tightlywith the pattern of close to dense packing. The polymerization mixtureis filled into the interstitial space of the large beads and polymerizedin these spaces. The flow pattern and column efficiency are improved bythe densely packed monodispersed beads. Materials with a very largenumber of or amount of separation-effective openings can be prepared inthis large diameter columns without fear of collapse of the media withlow rigidity since the large monodispersed beads are the supportingmaterials for the large columns. High flow rate can be achieved owing tothe large number or amount of separation-effective openings but robuststructure of the polymer.

In another embodiment, the heat dissipation problem is avoided inpreparation of the large columns with two or multiple stagedpolymerization incorporating polymers having separation-effectiveopenings as fillers. In one embodiment of the invention, multiple thincolumns having separation-effective openings prepared from the samepolymerization mixture are used as a filler to reduce the heatdissipation problem during in situ preparation of the large columns. Inanother embodiment of the invention, a polymer rod is used as the fillerfor the same purpose. In one embodiment of this invention, the fillermaterial is large non-porous silica beads.

The polymers, monomers, initiators, porogens, additives andpolymerization temperature are selected to improve the characteristicsof the column and may be used with an embodiment of polymerization usingpressure during polymerization or with other processes. Some of theaspects of this process may be applied to monomers and polymers formedin a polymerization reaction other than free radical reactions such asthe polycondensation reactions and sol gel process which form silicamonolith.

The column hardware in one embodiment of the invention includes rigidtubes to be used as chromatographic columns, with various shapesincluding cylindrical, conical, rectangular, and polygonal or anassembly of these tubes. The tube may be made from any conventionalmaterials know in the art including metal, glass, silica, plastic orother polymers, more preferably the stainless steel or glass. The innerdimension of this tube can be from micrometers to meters in diameter,thickness, width, or depth. The permeable solid material may span theentire cross-section area of the tube where the separation of thesamples take place by passing through the tube axially or radially (Lee,W-C, et al, “Radial Flow Affinity Chromatography for TrypsinPurification”, Protein Purification (book), ACS Symposium Series 427,Chapter 8, American Chemical Society, Washington, D.C., 1990.) dependingon the mode of separation, more specifically the axial or direct flowchromatography or the radial flow chromatography. The inner surface ofthe column or mold with which the polymerization solution is in contactduring polymerization may be non-reactive or may be treated to increaseadhesion to the surface of the plug. The tube can incorporate any usablefittings know in the art to connect it with other instruments, morespecifically chromatography instruments.

In an embodiment of this invention, the monolithic permeable solidpolymer is formed in a capillary tube, which can be for example acapillary tube with an internal diameter if 150 micons. In another, verysignificant, embodiment, the monolithic permeable rigid material isformed and sealed, often under pressure, in a removable 80. mm i.d.Teflon® sealing ring. This ring and column may be sold as a low-cost,reliable, high capacity, high resolution, very fast, easily replaceable,replacement chromatographic column. In another embodiment, the diameterof the tube is 10 mm. In another embodiment, the tube diameter is 4.6 mmand the material is stainless steel. In another embodiment of thisinvention, a plastic syringe barrel is used as a column. In anotherembodiment, the monolithic matrix is formed in a mold containing a metalcontainer, a sealing plate and an insert with multiple cylindricalholes. The thickness of the insert varies from 1 to 10 mm. A mold can bea micro device with plurality of channels or grooves on a plate made ofsilica or rigid polymers. The monolithic materials can be formed in anysizes and shapes make it suitable for a specially designed micro-sizeddevice, for example a micro-titer plates with multiple wells containingthe subject media and optionally having a small elution port in thebottom. There is no limit for the designed shape and size or theapplications with these devices.

The polymerization mixture is filled or injected into a column withdesired shape and size depending on the final use of the product to bepolymerized to form a plug having separation-effective opening for useas a solid support. Advantageously, the polymerization is done in acolumn in which the plug is to be used such as a chromatographic column,catalytic bed, extraction chamber or the like. In one embodiment of theinvention, the positive pressure is exerted to the polymerizationmixture during polymerization to control the particle size of theaggregates and to compensate for volume shrinkage during polymerization.The particle size of the aggregate has been found to be more homogeneousand larger than that from non-pressurized polymerization. The volumeshrinkage during polymerization is compensated by a positive airpressure or a moving piston with positive pressure.

More specifically, a polymerization mixture is applied to a column inthe preferred embodiment or to some other suitable mold. Polymerizationis initiated within the column or mold. The column or mold issufficiently sealed to avoid unplanned loss by evaporation of porogensor monomers if the polymerization is in an oven, or to avoidcontamination or dilution if polymerization is in a water bath. Duringpolymerization, pressure is applied to the polymerization solution.Preferably the pressure is maintained at a level above atmosphericpressure to control the size of the aggregates and its distribution inthe polymer, and to prevent the formation of voids on the polymer wallsurface and inside the media by shrinkage, and to prevent the media fromseparating from the wall of the column which forms alternative fluidpath through the gap or wall channels, until polymerization has beencompleted. Maintaining the column at atmospheric pressure to accommodateshrinkage did not prevent the formation of voids in every case andprovided poor reproducibility. The pressure source can be a gaspressure, a pressure from non-compatible liquid, a piston driven by airpressure, sprint force or hydraulic pressure.

In one embodiment of the invention any number of pressurized molds(tubes) can be kept at constant or controlled temperature in a singlewater bath, and identically pressurized from a single (e.g. nitrogen,water, etc.) manifold. This increases both uniformity and speed ofproduction.

In another embodiment of the invention, a selected pressure is exertedon the polymerization mixture by high pressure nitrogen. In stillanother embodiment of the invention, a selected pressure is exerted onthe polymerization mixture during polymerization by a pressurizationdevice shown in FIG. 2,3 or 4. The column is sealed in one end and thepolymerization mixture is filled into this column. The other end issealed by the device shown in FIG. 2,3 or 4. The whole assembly of thepolymerization fixture including the column is shown in FIG. 2,3 or 4.The pressure is applied to the polymerization by a piston with a smoothTeflon plug driven by a hydraulic pressure from a syringe pump. Thepolymerization mixture was sealed in the column by the Teflon plug andan O-ring. When a constant positive pressure is applied to thepolymerization mixture, the actual pressure is the difference betweenthe hydraulic pressure and the friction. During the polymerization, thepiston moves into the column upon the conversion of the monomers topolymers to compensate the voids generated due to the shrinkage of thepolymers. This prevents any negative pressure and void space generatedinside the sealed column due to this shrinkage thus improves the columnefficiency.

It is believed that the shrinkage is in every direction. The resultingvoids are probably occupied by the nitrogen gas generated by AIBN or bysolvent vapor with negative pressure inside the column. The voids can bea large irregular dents on the polymer wall or small irregular dentsspreading the entire polymer surface. The voids can also be distributedinside the polymer resulting in inhomogeneity of theseparation-effective opening size distribution. These irregular voidsand gaps result in the wall effect or zone spreading of the column. Theyare detrimental to the column efficiency and lower the resolution of thecolumn. These voids and gaps also result in low reproducibility of thecolumn performance from one to the other in the same batch of productionor from batch to batch of the productions.

In one embodiment of the invention, a selected pressure is exerted tothe polymerization mixture to control the size of the aggregates and theseparation-effective opening size distribution. The particle sizechanges with the change of the pressure on the polymerization mixtureduring polymerization. The particle size is larger at higher pressure.Under the positive pressure, the shrinkage of the polymer duringpolymerization happens only at the direction of the pressure force. Thisprevents the formation of voids inside the polymer and the voids/gaps onthe wall surface adjacent to the column wall.

During the polymerization process, the monomer concentration continuesto decrease with the increasing conversion of the monomers to polymers.The crosslinked polymers continue to precipitate out of the solution andaggregate with each other to form larger particles or clusters. Theseparticles precipitate and linked to each other by crosslinking agentssuch as an active polymer chain with a vinyl group. These interconnectedparticles sediment to the bottom of the column, which result in thelower monomer concentration at the top part of the column.

The separation-effective opening size is highly affected by the totalmonomer concentration and their ratios. An in-homogeneousseparation-effective opening size gradient is formed along the directionof gravity, which results in zone spreading. Since the particle size ispartly controlled by the pressure of polymerization, the gradient ofseparation-effective opening size can be corrected by adjusting thepressure during the polymerization. In one embodiment, the linearlyincreased pressure is exerted to the polymerization mixture duringpolymerization. In another preferred embodiment, the step pressuregradient is exerted to the polymerization mixture during polymerization.The speed and pattern of increasing/decreasing the pressure is chosen tocontrol the particle size of the aggregates and its distribution duringthe entire polymerization process. When a linear gradient ofseparation-effective opening size distribution is desired, it can alsobe achieved by changing the pressure during the polymerization withdifferent speed and different maximum pressure.

The polymerization temperature depends on the choice of initiator. WhenAIBN and Benzoyl Peroxide are used, the typical temperature range isfrom 50 to 90 degree C. The heating source can be any known in the art.The preferred ways are temperature controlled heating bath or oven. Thereaction time can be from 0.5 to 48 hours depending on the choice ofinitiator and reaction temperature. In one embodiment of this invention,the polymerization is carried out in a temperature controlled water bathat 60° C. for 20 hours.

Irradiation, such as IR, UV-vis or X-ray, is used as the source forpolymerization when light sensitive initiator is used. In one embodimentthe reaction starts by thermal activation of the initiator. In anotherit starts by the application of energetic radiation such as x-rays,either with or without a chemical initiator. If x-rays are used theinitiator should selected to thermally activate at temperatures wellabove, the polymerization temperature. On the other hand the initiatoractivates when under x-ray irradiation at temperatures in the givenregion desirable for the reaction mass to receive activation. Theinitiator is also selected so that activation time and temperature fordissociation is considerably less than for the monomers alone. Theproduction of active initiator (free radical) is controlled only by theX-rays intensity. Since the X-ray intensity is controllable, thereaction rate is controllable and won't “run away” or overheat.Moreover, the initiator may be chosen to activate when under X-rayirradiation at temperatures in the given region desirable for thereaction mass to receive activation.

In one embodiment, x-rays are used as the energy source forpolymerization. Energy of the x-ray photons are varied with thepreparation of the polymers with difference thickness or cross-sections.Lower energy x-ray is used for preparation of smaller diameter polymerrods and higher energy x-ray or exposure to lower energy x-ray for alonger period of time may be used for preparation of large diameterpolymer rods. In the preferred embodiment, the polymerizationtemperature is controlled by switching the x-ray on/off. When x-ray isswitched off, the polymerization is quickly shut off. within severalseconds since the lifetime of the free radical is typically around onesecond.

In one embodiment, photo initiator is used to initiate thepolymerization using x-ray as the energy source. The photo initiatorsare the typical photo initiators used in photo polymerizations- in thepolymer field including γ-ray, x-ray, UV, Visible and IR sensitivephotoinitiators. The photo initiators include azo compounds such asazobisisobutylonitrile, peroxides such as Diphenyl (2,4,6,-TrimethylBenzoyl) Phosphine Oxide, ketones such as phenanthrenequinone,2-chlorothioxanthen-9-one, 4,4′-bis(dimethylamino)benzophenone,4,4′-bis(diethylamino)benzophenone,4,4′-bis(dimethylamino)bezebophenone, 2-chlorothioxanthen-9-one, BenzilDimethyl ketal, Organic metallic complexes. These photo initiatorsinclude the ones used in both cationic and free radical polymerizations.In one embodiment, AIBN is used as the photo initiator. In anotherembodiment, phenanthrenequinone is used as the photo initiator.

In one embodiment, x-ray sensitizer or scintillator is used incombination with the photo initiator. The scintillators which can beused include all the luminescence materials in the prior art. Thescintillators include the compounds -containing benzene rings such asterphenyls, quarter phenyls, naphthalenes, anthracenes, compoundscontaining heterocycles, compounds with a carbonyl group, compounds withtwo or more lurorophors and organometallic compounds, and inorganiccompounds such as ZnTe, ZnSe, ZnS, Csl, Gd₂O₂S and CaWO₄. In someembodiments, 2,5-diphenyloxazole (PPO), 2-phenyl-5-(4-biphenylyl)1,3,4-oxadiazole (PBD), 2-(1-Naphthyl)-5-phenyloxazole (á-NPO are usedas scintillators. In one embodiment, terphenyl is used as thescintillator. In another embodiment, ZnSe is used as the scintillator.The mechanism of the initiation using the combination of scintillatorsand photoinitiators is believed to be a multiple step initiationprocess. First, the x-rays activate the solvent molecules to formelectronically excited solvent molecules. The excited solvent moleculesrapidly transfer their excitation energy to the scintillator formingelectronically excited scintillator. Then, the excited state of thescintillator relax to ground state by emission of photons. The emittedphotons were absorbed by the photo initiators and forming active freeradicals. The free initiator free radicals contact the vinyl functiongroups of the monomers and start the polymerization process. The processcan be depicted as followings:

-   -   X-ray hγ—excited solvent molecule*—excited scintillator        molecules*—UV or Visible hγ′—excited initiator        molecules*—initiator free radicals-polymer radicals

x-ray irradiated polymerization can be used for preparing homopolymerssuch as polystyrene, polytetrahydrofuran, resins such epoxy or othercrosslinked materials, and porous polymer support such as separationmedia in the shape of columns, membranes, or materials in any otherhousings. The method can be used for preparation of molded parts in anyshape. In one embodiment, polystyrene is prepared by using x-rayirradiation as energy source. Polystyrene is prepared in a mold of aglass vial with narrow opening and in a shape of a cylinder. Bothsolution polymerization and bulk polymerization are used to preparehomopolystyrene materials. In another embodiment,polyglycidylmethacrylate resin is prepared by using the same method. Inanother embodiment, porous poly(styrene-co-divinylbenzene) is prepared.In another embodiment, porous poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) polymer support is prepared.

X-rays can be used for preparing porous monolithic polymer supports withcross sections from micrometers to meters, and the particle-shapedpolymer supports as well. X-ray irradiated polymerizations are used toprepared monolithic liquid chromatography columns including capillaryand microbore analytical columns, conventional analytical columns, andpreparative and process columns, In one embodiment, porous monolithicsupport in a glass column with inner diameter of 1 cm is prepared. Thecolumn is characterized by SEM, porosimetry and chromatography. Thepoly(styrene-co-divinylbenzene) monolithic column in this glass housingshows excellent separation of peptides. X-rays can penetrate thematerials in depth. Both organic and inorganic polymers can be preparedusing x-ray or γ-ray. High energy x-ray and γ-ray can travel thematerials in high depth. If a greater such depth is required, theunpolymerized mixture can be placed in the x-ray beam, and rotated andtranslated in a way chosen that the overall, time-averaged irradiationis sufficiently uniform throughout the body being polymerized. Note thatradiation aligned in the intended direction of chromatographic flow doesnot cause significant chromatographic nonuniformity in the bed becauseof absorbance of the radiation beam. Axial nonuniformities do notnecessarily degrade performance as radial nonuniformities do. Low energyx-ray penetrates the materials in less depth but is safer to use. In oneembodiment, low energy x-ray was used to prepare a porous monolithicsupport in a glass column with the diameter of 3.5 cm. In anotherembodiment, a monolithic support in a polymeric columns housing with 88mm diameter is prepared. The temperature of polymerization is controlledby controlling the intensities and energies of the x-ray photons and bya water jacket so at least one side of the reaction mixture. Preferablysuch water cooling cools the column in its axial direction but not theradial direction to avoid thermal gradient and resulting inhomogeneity.All flow paths experience the same inhomogenuity in the direction ofchromatographic flow, and thus have no effect upon the separation. Thepolymerization temperature in the center of this large diameter columnis about the same as the polymerization temperature on the edge of thecolumn during the whole polymerization process. The conversions of themonomers are completed after 4 days of polymerization.

X-ray irradiated polymerization can be combined with thermalpolymerization. Polymerization rate and porosities can be controlled bythe rate of polymerization, which can be controlled by the energies andintensities of x-ray, and the temperature of polymerization. Inconsideration of the economy and safety of irradiated polymerizationprocess, lower energy and intensity x-ray is preferably used inpreparation of large diameter monolithic polymer support. The conversionof monomers is more than 70% complete after 48 hours of polymerizationin the 3.5 cm diameter column. In order to speed up the polymerizationrate and finish the conversion in a short time, thermal polymerizationat desired temperature corresponding to the initiators is used. In oneembodiment, porous poly(styrene-co-divinylbenzene) is prepared by usingx-ray irradiation followed by thermal polymerization at 70° C. after thex-ray irradiated polymerization. The porosity of the polymer can becontrolled by monomer compositions, x-ray intensity and energy, choiceof initiator and scintillator combination, and the polymerizationtemperature in both x-ray irradiated polymerization and thermalpolymerization. The porogenic solvents used in this polymerizationprocess is the same as those used in pure thermal polymerizationdescribed earlier. Excellent polymer support is obtained for liquidchromatography use.

Ultraviolet and visible light radiation has been used in the past forpreparing polymers including homopolymers, slab-shaped polymer resinsand porous polymer surface coatings but not the polymer materials inthis invention. Both UV and visible light can penetrate thin, solid,clear materials. Ultraviolet and visible lights have been used toprepare thin, clear materials such as membranes. The homogeneity of aporous polymer becomes more of a problem when the thickness of materialsincreases. This is due to the scattering and attenuation of lightsthrough the porous solid materials. The depth that the light can travelthrough the solid materials decreases quickly depending on thefunctional groups and the refractive indexes of the materials. Consideran acrylic, dry porous column, made of two phases: acrylic resinrefractive index about 1.5 and air with refractive index of 1.00. Thisscatters light so completely that this material looks like chalk, whiteand opaque. Heretofore then these apparent differences have preventedthe use of UV or visible light for polymerization. This patentapplication discloses a method of using ultraviolet and visible lightsto prepare polymer materials including materials listed above with highhomogeneity. This patent application also discloses a method ofpreparing polymer materials many inches thick.

In this invention, a solvent of very similar or the same refractiveindex to the targeted polymer resins at the selected wavelength of lightis chosen for the polymerizations. The lights at the selected wavelengthcan travel though the polymers during the polymerization due to thelittle used Christiansen Effect, wherein the targeted porous resinbecomes quite translucent or transparent. Light in a particularwavelength range show transparency because the pores are full ofsolvent. Preferably, monochromatic light, of which the indices of therefraction in both the polymer solid and solvents are very close, ischosen for the polymerization. An initiator activated by light ratherthan heat is used with a photoinitiator. If white light is used, thetransmitted light corresponds to the particular wavelength at which therefractive indexes of the light in both polymer solid and the solvent orsolvent combinations are very close. A photo initiator is chosen toabsorb and be activated by light in the near transparent polymer andsolvent wavelength range. The initiator absorbs this wavelength range.Preferably, when a photoinitiator is activated it breaks up ontofragments lacking in the original chromophores. Such fragments have noabsorption in the range of selected wavelength of the light. When alight ray is absorbed by the chromophore in a photo initiating moleculethe result is local polymerization at the surface. If the chromophoressurvive this reaction they will be there to block the next ray of lightfrom activating the next photo initiator molecule, hence limitingpolymerization to the original surface. Therefore, the light can travelthrough the polymer mixture in more depth and the transmission of thelight increases. This effect is known as Bleaching Effect. It has beenfound that a good photo initiator in this respect is2,4,6-trimethylbenzoyidiphenylphosphine oxide. The bleaching effect andthe Christiansen effect are synergistic in the production of very thicklayer, or deep, porous polymers by photoinitiation or e-ray initiation.In the latter case x-rays excite the solvent which then transmitssecondary energy to a so-called “sensitizer” which then fluoresces toactivate the initiator. It can also be seen that if the solvent whenexcited by x-ray also in itself fluoresces, an additional degree ofsynergy exists. However, often the use of p-terphenyl as a sensitizerfor x-ray polymerizations. Fluorescing solvents that are used includemany aryl compounds such as toluene, o-, m-, or p-xylene, or the 1,2,3(or other isomers of) mestylene. In one embodiment, homo-poly(glycidylmethacrylate) is prepared by ordinary solution polymerization usingo-xylene as the porogenic solvent. The refractive indexes of 0-xylene,the monomer and especially the polymer are close. The resulted polymerin solution is close to transparent but a little translucent due tominor scattering of the white light.

The refractive indexes of target polymers are measured. Both the goodand poor solvents are selected to have the refractive indexes close tothe refractive indexes of the target polymers. By tuning the ratio ofthe solvents, the refractive indexes of the solvent mixture will bealmost the same as the polymer. Therefore, the transmission of the lightthrough the polymer swelled by the solvents can reach the maximum. Inone embodiment porous polymethacrylate based monolith is prepared byusing the combination of solvents.

The resulting permeable polymer may have more than one suitableconfiguration. One has a desired separation-effective opening sizedistribution for target applications. In general, it includes smallseparation openings less than 300 nm in at least one direction whichprovide high surface area for separation, and large separation openingssuch as larger than 500 nm for the majority mobile phase to go through.Preferably the sizes of large separation-effective opening are between 2to 5 microns for medium and low pressure separation media, and 0.6 to 2microns for high and medium pressure separation. Theseparation-effective opening size distribution and the irregular featuresize distribution can be controlled by the types and amount of porogens,monomers, initiators and polymerization temperature, time and pressure.In one embodiment, the monomers are selected not only to have desiredfunctionality, but also to help improve the column efficiency bychanging the kinetics of polymerization and polymer structure, whichleads to more ideal separation-effective opening size distribution. Theratio of monomers is selected for the same purpose. The type and amountof porogens are selected after careful investigation for the generationof the desired separation-effective opening size distribution. The useand selection of pressure during polymerization is particularlyimportant for the generation of the desired separation-effective openingsize distribution and the homogeneity of the separation-effectiveopening size distribution through out the whole column. It also preventsthe formation of irregular voids and wall channels which happened inconventional sealed or open polymerization.

It has been found that the formation of micropores can be drasticallyreduced or prevented by using pressure during polymerization and thecareful tuning of the other polymerization conditions and reagents. FromScanning Electron Microscopy (SEM) studies of this polymer havingseparation-effective openings, it is found that the polymer particlemorphology is similar to some form of coral. The coral-like polymer isformed of interconnected corrugated particles which apparently havegrown by accretion. The interior of these particles are non-porous. Thesurface is highly corrugated and contains huge number of small openshallow grooves with various sizes of openings. In SEM study of one ofthe polymers the particles are core-shell structured with short depth ofthe openings. These unusual corrugated polymer structures and core-shellparticle structures without through pores in the individual particlesgreatly improve the capacity of the monolith compared to regularnon-porous separation media while avoiding the mass transfer problem inconventional macroporous media containing a large number of microporesand mesopores inside the particle or beads. These structures are alsodramatically different from so called “Perfusion Beads” or monolithswith though pores disclosed in the literatures. The aggregates ofparticles in some columns conditioned by the application of pressure orby holding the volume against expansion when internal forces tend toswell or expand the polymer results in stacked generally flat or nestedconfiguration rather than the aggregated substantially round aggregatesof particles.

The monolith in this invention combines the advantages of highresolution in non-porous particle packing and the high capacity ofmacroporous packing while avoiding their problems. These corrugatedparticles may grow by accretion from polymer nucleus which are swelledand surrounded by monomers and active oligomers, and merging with otherpolymer nucleus. These particles aggregate with each other and arereinforced by crosslinking. This structure improves the columnefficiency greatly by prevention of the trapping of sample molecules inthe micropores, and in the pores inside the particles, which are one ofthe major reasons for zone spreading according to the theoretical model.The size of the particles, aggregates or clusters can be finely tuned tobe more homogeneous, and the separation-effective opening sizedistribution can be improved to give high resolution separation bycareful control of pressure in combining with the selections of otherfactors discussed earlier.

Satisfactory separation of more hydrophilic compounds in liquidchromatography often requires the starting mobile phase to be 100% waterwith no organic solvent. This type of separation can not be achieved toa satisfactory extent with reversed phase media based on purestyrene/divinylbenzene, polymethacrylates and their derivativescontaining C4, C8, C12 and C18, which are very hydrophobic. The polymersshrink in aqueous mobile phase with low organic solvent contents. Theshrinkage results in void space between the column wall and the polymermedia, which leads to so called “wall channeling” in chromatography. Asthe consequence, the sample and mobile phase bypass the media and gothrough wall channels instead of the media. The sample is not retainedor partly retained which results in multiple peaks for each pure sample.

In one embodiment, wall effect is avoided by first shrinking the mediadown to the maximum extent by passing pure water or salt solutionthrough the media, and then followed by compression of the media withpiston fittings until the void space between the wall and polymer mediais sealed. The piston is held in place by a nut fitting. The fittingsare shown in FIGS. 3 and 4. The shrinkage and sealing of the wall voidcan be monitored by first decreasing then increasing the back pressureof the column. This process prevents the formation of the “wallchanneling” during the separation process. In one embodiment, thepolymer is shrunk in pure water and compressed with PEEK pistonfittings. In another embodiment of this invention, the polymer is shrunkin 1 mol/I NaCl solution and compressed with PEEK piston fittings.

In highly polar environments, the linear polymer chain, and the chainsof C4, C8, C12, C18 on the polymer surface collapse to the surfaceresulting in poor interaction between the sample molecules and thesurface of the media. Also, the bed is very poorly wetted by the mobilephase due to the extreme difference in polarity of the media and mobilephase. The mass transport and interactions between the sample andstationary phase is very poor, which result in low column efficiency andresolution. These problems and the problem of “wall channeling” arereduced by increasing the hydrophilicity of the polymer matrix whilemaintaining the desired hydrophobicity for sample retention andseparation.

The polymer matrix contains hydrophilic functional groups such ashydroxyl, amide, carbamide or hydrophilic moieties in the polymerrepeating units. The polymer can be wetted and swelled by water due tothe hydrophilicity of the polymer matrix. The surface of the polymermedia still contain highly hydrophobic polystyrene chain, or C4, C8, C12and C18 chains for the hydrophobic interation in reversed phasechromatography. The shrinkage of the polymer in water is reduced orcompletely prevented, which also solve the problem of wall channeling.The hydrophilicity can be improved by direct copolymerization ofmonomers containing hydrophilicity moieties, or by modification ofpolymers to incorporate the hydrophilic moieties.

In one embodiment of this invention, hydrophilic hydroxylethylmethacrylate is copolymerized with styrene and divinylbenzene for theprevention of “wall channeling” and collapse of the hydrophobicinteraction chains in water. In one embodiment of this invention,stearyl acrylamide is copolymerized with stearyl methacrylate andethylene glycol dimethacrylate. The addition of hydrophilic monomersalso may decrease protein denaturation in reversed phase columns. In onepreferred embodiment, acrylonitrile is copolymerized with styrene anddivinylbenzene. The polymers swell more and more with the increase ofthe hydrophilicity of the hydrophilic monomers. The reverse phaseseparation media constructed with the polymer containing hydrophilicmoieties will swell in both aqueous and non-aqueous solutions. Thisenlarges the applicability of reversed phase separation media in aqueousmobile phase.

In Situ polymerization can be used to prepare the columns with any sizesand shapes. In one embodiment, a capillary column with cylindrical shapeof inner diameter of 75 μm was prepared. In another embodiment, a columnwith 4.6 mm ID was prepared. In another embodiment, a column with 88 mmID was prepared. In another embodiment, a square capillary column withcross-section of 100 μm and on up to 700 μm was prepared. In anotherembodiment, a polymer disk with 3 mm thickness and 1 cm inner diameterwas prepared. In another embodiment, a donut shape monolith with 1 cmouter diameter and 4.6 mm inner diameter was prepared. In anotherembodiment, a microchip column with 100 μm inner diameter grooves wasprepared.

Hydrophobic interaction chromatography requires very hydrophilicseparation media with mild hydrophobicity. Upon further increase of thehydrophilicity of the matrix with less hydrophobic carbon chain orpolymer chain, the reversed phase media can be turned into hydrophobicinteraction media.

Normal phase chromatography requires hydrophilic media, whose surface isfully covered by hydrophilic functional groups. Hydrolysis of the epoxygroup in poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)was used in prior art to obtain normal phase separation media. TheNormal Phase separation media is prepared by in situ directpolymerization of hydrophilic monomers containing hydrophilic functionalgroups such as hydroxyl and amide. In one embodiment hydroxylethylmethacrylate is copolymerized with a crosslinker, such as EDMA, toobtain normal phase media. In one embodiment of this invention, thecolumn hardware is a polypropylene barrel reinforced with glass fibers.

The reversed phase monolithic media prepared according to prior art hasextremely low capacity and is compressed during separation. The loadingcapacity of the liquid chromatography media and the rigidity of themedia is increased by increasing the crosslinking density of the media.The crosslinking density is increased by using higher amount ofcrosslinker, such as divinylbenzene, in poly(styrene-co-divinylbenzene)monolith. In one embodiment of this invention, 100% of divinylbenzene(80% purity. The rest of them are mostly ethylstyrene. It is the highestpurity grade available commercially.) in total monomer is used. Thecapacity is six times higher than the monolith prepared in the priorart. The high capacity monolith prepared under pressurizedpolymerization has high resolution as well as high capacity. In oneembodiment of this invention, 90% of the divinylbenzene (80% purity) intotal monomer is used. In another embodiment, 80% of the divinylbenzene(80% purity) in total monomer is used.

Another method of improving the rigidity and resolution of the media isby increasing the total polymer density in the column. The total polymercontent is increased by increasing the total monomer content in themixture. By increasing the total monomer content in the polymerizationmixture, the resolution of column is improved as well. Theseparation-effective opening size and its distribution are highlyaffected by the total monomer concentration in the polymerizationmixture. In one embodiment of this invention, 46% weight percent oftotal monomers is used to improve the rigidity and resolution of themedia.

The monolithic media prepared in the prior art has poor resolution, lowspeed of separation, low rigidity and extremely low capacity. Theprior-art monolithic polymethacrylate based weak anion exchanger wasprepared by modification of poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) with neat diethylamine. It swells extensively inwater and can not be used at high flow velocity. This medium has verylow rigidity and is not stable at flow velocities more than 6 cm/min.The back pressure of the column keeps increasing during the runs. Twomethods of improving the rigidity of the hydrophilic medium areprovided.

First, the rigidity of the medium can be improved by increasing thecrosslinking density of the polymer matrix. At low crosslinking density,there are a lot of non-cross linked linear polymer chains which aresolvated by water and extend out to the solvents. The polymer matrixexpands due to this extensive solvation. The expansion of polymer matrixin a polymer narrows the size of the separation-effective openings andinterstitial spaces between the interconnected particles. The porosityis also decreased. These result in high back pressure. The highlysolvated porous polymer has characteristics of soft gel. Under apressure, the soft polymer can be compressed easily and leads to higherpressure. The increase of pressure will further compress the medium andlead to even higher pressure. The cycle of pressure increase andcompression make the prior art monolith not useful for the applicationin high speed separation.

Upon the increase of crosslinking density, the swellable linear polymerchains are shortened and the swelling is reduced. The polymer withseparation-effective openings becomes more rigid. The structure withseparation effective openings is maintained in aqueous solvents. Highporosity and larger separation-effective opening size can be obtainedwith highly crosslinked polymer matrix, which allows higher flow rate tobe used without the detrimental cycles of the compression and pressureincrease. High speed separation can be achieved by using high flow rate.

In one embodiment of this invention, the cross linking density of theion exchanger is greatly improved by using 70% crosslinker, ethyleneglycol dimethacryalte (EDMA). In a preferred embodiment, the amount ofEDMA is 50%. In another preferred embodiment, the amount of EDMA is 60%.EDMA is more hydrophobic than its copolymers containing ion exchangegroups. By increasing the amount of EDMA, the hydrophilicity of thepolymer matrix is reduced. This results in decrease of swelling andimproves the rigidity. This is the additional advantage from using morehydrophobic cross linker instead of hydrophilic crosslinker when highrigidity of the polymer in aqueous phase is highly desired.

Second, the rigidity and column efficiency of the polymer separationmedia are both improved by controlled modification in this invention.The glycidyl methacrylate (GMA) is hydrophobic before it is modified tocontain ion exchange functional groups. The GMA in prior art monolithicweak anion exchanger (WAX) is modified by reaction with neatdiethylamine at 60° C. for 3 hours. Neat diethylamine can swell thepolymer and diffuse into the polymer particles to access the GMA epoxidegroups. This modification reaction modifies not only the GMA moieties onthe separation-effective opening surface of the polymer but also thoseinside the polymer matrix. The hydrophilic moieties containing the ionexchange functional groups intermingle with hydrophobic backbone bothinside and outside the separation-effective openings after modification.This non-selective modification makes the whole polymer matrix swellextensively in water while some hydrophobic backbones are exposed on thesurface of the separation effective openings. The hydrophobic patches onthe surface of the separation openings can result in secondaryhydrophobic interaction during ion exchange chromatography separations,which leads to zone-broadening.

A controlled modification of the GMA on the surface of the polymerparticles can keep the internal part of the particle more hydrophobicand less swellable in water. After modification of the surface GMA, thesurfaces of the particles become much more hydrophilic and attract thewater molecules. Those more hydrophobic backbones retreat to inner partof the polymer particles to stay with more hydrophobic cores of theparticles, and get away from the very polar buffer environment duringchromatography separation. This increases the coverage of surface withhydrophilic ion exchange groups and prevents the zone-broadening fromsecondary hydrophobic interaction. The controlled surface modificationis accomplished by catalyzed modification reaction in aqueous solutionat lower temperature. The catalyst is preferably an acid or reagentwhich can generate protons in situ.

In the preferred embodiments of this invention, a dialkyl amine hydrogenchloride salt is used as a catalyst. The salt solution is very polar andhas less tendency to swell the hydrophobic polymer. The ionic catalysttends to stay in solution instead of diffusing into the veryhydrodrophobic internal matrix. The lower reaction temperature reducesthe swellability of the polymer. Diethyl amine might diffuse into thepolymer matrix but the reaction of diethyl amine with GMA at lowtemperature is very slow and insignificant. In one embodiment of thisinvention, the dialkyl amine hydrogen chloride catalyst is diethyl aminehydrogen chloride. In another embodiment of this invention, the catalystis trimethyl amine hydrogen chloride. In one embodiment of thisinvention, the reaction temperature is 25° C. In another embodiment ofthe invention, the modification temperature is 30° C.

The prior art processes for the synthesis of monolithic weak cationexchangers are based on membrane, beads and gels and none of them can beused directly in the in situ preparation process of monolithic columns.Seven methods for preparing a weak cation exchanger a described below.All the modification reactions in these methods are carried out bypumping the modification solution through the column continuously at aselected temperature, or the reagents are sealed inside the column andheated in a heating bath or oven after the reagents are pumped into thecolumn.

The first method is the two-step modification of poly(glycidylmethacrylate-co-ethylene glycol dimethacrylate) (PGMAEDMA) withchloroacetate salt. Sodium chloroacetate has been used to modifyhydroxylethyl methacrylate based material to obtain carboxylic acidgroups in the literature. In order to take advantage of the abovereaction for the modification of monolith, the epoxide ring in GMA isfirst opened to obtain hydroxyl group by hydrolysis using 1 M H₂SO₄aqueous solution. In the second step, chloroacetate couples with thehydroxyl group in the polymer to attach the carboxylic group to thepolymer. This reaction is catalyzed by strong base such as sodiumhydroxide. The reaction temperature is from 40 to 80° C., preferablyfrom 50 to 70° C. The reaction time is varied from 1 to 24 hours,preferably less than six hours. In one embodiment of this invention, themodification reaction takes place by pumping 5 M sodium hydroxideaqueous solution through the monolithic column at 60° C. for 2 hours.The capacity of this media is not ideal although the column efficiencyis good. The capacity can be increased by longer reaction and higherreaction temperature. However, the separation medium becomes soft due tothe side hydrolysis reaction of the esters in the polymer. Thecrosslinking density is lowered since the crosslinker EDMA is hydrolyzedas well.

The second method is a one-step modification of the GMA in the polymerto obtain the carboxylic functional groups using glycolic acid asreagent. Glycolic acid is reacted with PGMEDMA at temperature between 40to 90° C. for 1 to 24 hours. This reaction is a self catalyzed reactionsince glycolic acid is a catalyst itself. The reaction can be catalyzedby other stronger acid such trifluroacetic acid (TFA). The reaction issimple but the capacity of the weak cation exchanger is low due to aparrarel side reaction. The epoxide ring can be opened by hydrolysisreactions as the side reaction. In order to prevent this reaction, thenon aqueous solvent is used. Preferably, solvent containing protons isused. In one embodiment of this invention, glycolic acid solution informic acid containing TFA catalyst is pumped through the column for 3hours at 80° C.

The third method is a double modification of the PGMEDMA with bothglycolic acid and choroacetate. There are several advantages of thedouble modification reactions. First, they can all be performed inaqueous solution; Second, both reaction steps lead to desired product;Third, the side reaction of the first step leads to the desiredfunctional group for the second step modification; Fourth, theconditions of double modification reaction can be milder than the singlereaction to obtain the same or higher capacity while avoiding thehydrolysis of the backbone which maintains the rigidity of the matrix.In one embodiment of this invention, the first reaction is performed inglycolic acid aqueous solution containing TFA as catalyst, and thesecond reaction is the substitution reaction of chloroacetate by NaOHaqueous solution.

The fourth method is a one-pot reaction of glycolic acid andchloroacetate. Instead of the double sequential reactions, both reagentsare put into the solution together during reaction. The reaction withglycolic acid is base-catalyzed instead of acid-catalyzed. This methodhas the the advantage of the third method but with lower capacity due toless reactivity of the base-catalyzed ring-opening reactions by glycolicacid in water.

The fifth method is a hydrolysis reaction of acrylates or methacrylates.The hydrolysis of the ester groups leads to the carboxylic functionalgroups. The direct hydrolysis of PGMEDMA membrane or beads is known inthe prior art. However, the resulting media did not have either goodcapacity or separation. It is discovered in our work that both theresolution and capacity can be dramatically improved by hybriding thehydrophilic and hydrophobic acrylates or methacrylates. The hydrolysisreaction is much more efficient since the water molecule can diffuseinto the surface of the particles and wet the surface much better due tothe hydrophilic moieties of the acrylates. The reaction can be catalyzedby both acid and base, such as TFA or NaOH aqueous solution. In oneembodiment of this invention, poly(methyl methacrylate-co-hydroxylethylmethacrylate-co-ethylene glycol diemthacrylate) is prepared andhydrolyzed to obtain weak cation exchanger. In another embodiment ofthis invention, poly(hydroxylethyl methacrylate-co-ethylene glycoldimethacrylate) is hydrolyzed. In another embodiment of this invention,PGMEDMA is hydrolyzed by acid first and base in the second step. Theweak cation exchanger obtained in this method is softer due to thehydrolysis of the backbone crosslinker.

The sixth method is a direct copolymerization of acrylic or methacrylicacid. The direct copolymerization of the acid leads to the weak cationexchanger in one step. This method greatly simplifies the preparationmethod. The capacity of the weak cation exchanger is relatively higherthan the modification method but still not ideal. The ratio of theacidic monomer to crosslinking monomer is between 2% to 30%, preferably5% to 15%. With the higher content of the acid monomer, the capacity ishigher but the media is softer. The direct polymerization method isapplicable to the preparation of monolithic membranes, columns, chips,tubes or any format known in the art.

The seventh method is the combination of direct copolymerization and thecontrolled modification. It was discovered in this invention that thiscombination leads to high capacity while maintains the rigidity of themedia. The resulting media can be used for high-throughput separationusing high flow velocity. The improved capacity by direct polymerizationof acrylic or methacrylic acid reaches a limit due to the softness ofthe media containing high amount of the acid. The acidic monomers arerandomly polymerized and dispersed throughout these matrixes. Theseacids are converted to salts in buffer and resulted in extensiveswelling of the media in aqueous mobile phase. Also, the hydrophobicbackbone consists of carbon chains and esters are exposed on the surfaceresulting in secondary hydrophobic interaction during ion exchangechromatography separations. This leads to zone-broadening and tailing.The hydrophobic surface can be further modified to become hydrophilicwhile improving the capacity. The controlled modification improves thecapacity and hydrophilicity of the media while preventing the softnessof the media. Over modified media will leads to the modification insidethe particles besides the modification on the surface. As discussed inthe preparation of weak anion exchanger, the modification inside theparticle results in extensive swelling which blocks theseparation-effective opening or changing of the morphology which leadsto detrimental cycle of pressure increase and compression. In oneembodiment of this invention, weak cation exchanger is prepared bycopolymerization of acrylic acid, methyl methacrylate (MMA) and EDMA inthe first step, and hydrolysis of the methyl methacrylate in the secondstep. The hydrolysis of MMA is base-catalyzed and accelerated by thepresence of very hydrophilic acrylate salt, which is the conversionproduct of the acrylic acid after reaction with NaOH.

This methodology of combining direct polymerization and modification isapplicable to the preparation of all hydrophilic polymer supports whichrequire high number of functional groups and rigidity of the matrix atthe same time. It is applicable in preparation of monolithic tubes orcolumns, monolithic membranes, monolithic capillary and chips or anyother monolith in different format, as well as polymeric particles,gels, membranes or any other type of polymeric separation media. Inparticular, the resolution and capacity of monolithic membrane can beimproved with this method. The improvement can be achieved by in situprocess of by off-line process. The monoliths or beads obtained bydirect polymerization can be modified by pumping the reaction solutionthrough the packed columns or membranes, or immerge them into themodification solution. The beads can be suspended in the modificationsolution. By using this methodology, we developed high capacity stronganion exchanger (SAX) and strong cation exchanger (SCX).

The prior art processes for making monolithic strong anion exchangersare based on membrane, beads and gels and are not applicable to themanufacture of monolithic strong anion exchange column. Three methods ofpreparing monolithic strong anion exchange column in situ are providedbelow.

Method one is the combination of highly rigid polymer and controlledmodification of the surface. Modification of PGMEDMA with trimethylaminehydrogen chloride has been used to obtained membrane and bead basedstrong anion exchanger. When the reaction is used for preparation ofmonolithic strong anion exchangers, the resulting media is soft and cannot be used for high speed separation. The rigidity is improved in twoways as in the preparation of monolithic weak anion exchanger in thisinvention: High crosslinking density and Controlled Modificationreaction.

The basic polymer for SAX is formulated to contain high crosslinkingdensity by using higher ratio of crosslinking monomer to functionalmonomer. The amount of crosslinker is increased to more than 50% of thetotal monomers in the polymer. In one embodiment of this invention, 60%EDMA in total monomers is used. The porogens and their ratios areselected to offer optimal resolution at relatively low pressure.

The controlled modification is accomplished by catalyzed amination ofthe PGMEDMA. The catalyst can be any base known in the art. In oneembodiment of this invention, trimethyl amine is used as catalyst. Theamount of the catalyst is from 1% to 50% volume of the solution,preferably between 10% and 30%. The reaction temperature is between 10to 60° C., preferably between 20 to 50° C. The reaction time is between10 minutes to 24 hours, preferably between 1 to 4 hours. The selectedcatalytic reaction modifies the surface of the particles more than theinternal part of the particles, which results in the media to be used athigh flow rate. In one embodiment of this invention, the reaction iscarried out at 40° C. for 3 hours.

Method two is the direct copolymerization of monomers containing thequarternary amine, or their intermediate which can generate thequaternary amine in situ. In one embodiment of this invention, thefunctional monomer containing quaternary amine is 2-(acryloyloxyethyl]trimethylammonium methyl sulfate (ATMS). The polymer has highcrosslinking density. The ratio of the crosslinking monomer in the totalmonomers is between 50% to 70%. In one embodiment of this invention, 60%EDMA is used. The amount of ATMS is between 2% to 20%, preferablybetween 5% to 15%. The third monomer which makes up the rest of monomeris preferred to be hydrophilic monomers such as HEMA althoughhydrophobic monomer can be used as well.

Method three is the combination of direct polymerization and controlledsurface modification shown in method two and one. The strong anionexchanger obtained by method one is rigid and have high resolution.However, it suffers from non-ideal capacity. Method two improves thecapacity but not sufficient and suffers from lower resolution. Thecombination of direct polymerization and controlled surface modificationdoubles the capacity and improves the resolution and recovery. Therecovery of proteins is improved since the surface is fully covered byhydrophilic protein benign groups. Secondary hydrophobic interaction,which is the main reason for lower protein recovery, is minimized. Theporogens are researched and selected to offer the desired flow rate. Inone preferred embodiment of this invention, the combination ofbutanediol, propanol and water is used as porogens. The polymerizationmixture and conditions are formulated to offer the optimal resolution atthe desired flow rate.

Prior art processes for preparing monolithic strong cation exchangersare based on membrane, beads and gels and are not transferable to the insitu preparation of monolithic strong cation exchange columns. Threemethods for preparing monolithic strong cation exchange columns in situare provided below.

Method one is the modification of PGMEDMA with butane sultone or propanesultone catalyzed by strong base soluble in organic solvent.Modification of PGMEDMA with propane sultone using NaOH solution as thecatalyst has been used to prepared membrane or bead-based strong cationexchanger. The reaction was a two-phase reaction since propane sultoneis not soluble in NaOH aqueous solution. The two-phase reaction mixturecan not be pumped through the column to carry out the modification.Several approaches have been taken to carry out the modificationreaction. Approach one is a two-step modification reactions consist ofactivation of the media with strong base such as potassium t-butoxide inthe first step followed by nucleophilic ring-opening reaction withbutane sultone. Butane sultone is preferred since it is a liquid butpropane sultone is a solid at room temperature. Potassium t-butoxide ispreferred since it has higher solubility than it sodium counterpart. Thesolvent is a good solvent of the reagent such as dimethylsulfone (DMSO).The modification solution has to be homogeneous in order to be pumpedthrough the column for in situ modification. Approach two is a one-potreaction consist of both activation and modification steps. Both strongbase and butane sultone are dissolved in a strong solvent. The solutionis pumped through the column continuously or sealed at a selectedtemperature for several hours. The reaction temperature is preferablybetween 80 and 120° C. In one embodiment of this invention, 90° C. isused. In another embodiment of this invention, 120° C is used.

Method two is a direct polymerization of monomers containing strongcation exchange group. In the preferred embodiment of this invention,2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) is used as thefunctional monomer containing sulfonic group. The amount of AMPS isbetween 2% to 20%, preferably 5% to 15%. The capacity of this polymer isgreatly improved comparing to the first method. The polymerizationmixture is formulated to offer the optimal resolution at desired flowrate.

Method three is the combination of direct polymerization and controlledmodification. In one embodiment of this invention, AMPS is copolymerizedwith GMA and EDMA. The polymer is further modified by controlledmodification as described in method 1. Both the capacity-and resolutionis greatly improved comparing to the method 1 and 2. The amount of EDMAis preferably to be between 50% to 70%. The amount of AMPS is preferablybetween 2% to 15%. The rest of the monomer is GMA. In another embodimentof this invention, the AMPS is copolymerized with HEMA and EDMA. Theporogens are researched and selected to offer the desired flow rate. Thepolymerization mixture and conditions are improved to offer highresolution and high speed chromatography.

Preparation of large diameter monolithic columns for effectivechromatography separation by in situ polymerization method has been avery difficult task due to the heat isolating effect of polymer formedby exothermic polymerization. It was found that the heat transfer isfast enough to prevent the inhomogeneity of the polymerizationtemperature as long as the shortest distance (defined as radius in thiswriting) between the center of the monolith to the surface of themonolith is less than 8 mm depending on the materials of columnhardware.

To prepare large diameter monolithic columns, with reduced heat ofpolymerization problems, multiple staged polymerizations are used. Theresulting polymer monoliths in each stage of polymerization have radiusup to 8 mm if the mold for polymerization is made of good heattransferring material. This is accomplished by first preparing columnswith radius less than 8 mm and using them as fillers for the secondstage polymerization, in which the radius of the polymerizationsolutions between the fillers and the column wall is also less than 8mm, and the distance between the fillers is less than 2 mm. It is foundin this work that the thickness of the polymerization solution betweenthe fillers less than 2 mm has insignificant effect on the variations ofthe polymerization temperature. Multiple thin polymer columns are filledinto a large diameter column and filled with the second stagepolymerization mixture. The column is sealed with regular fittings orfittings to allow pressurization during polymerization. The largediameter column is then placed into a temperature controlled heatingbath or oven to carry out the second stage polymerization.

The thin columns are prepared by the process disclosed above withpressurized or non-pressurized polymerization. The thin monolithiccolumns prepared in the first stage polymerization are preferablypreserved without washing and further modification. A polymerizationmixture for the second stage polymerization is the same or differentfrom the polymerization mixture of the first stage polymerizationdepending on the types of media. The thin columns with radius less than8 mm can be solid rods, discs, hollow tubes with thickness of thecylinder wall less than 8 mm, or a membrane. The shapes of the abovethin columns can be any shape known in the art, such as round,rectangular, triangle, etc. It is perceivable that the fillers can beother particles described in the section of filler materials in thisspecification.

In one embodiment of this invention, multiple thin columns with radiusof 5 mm are used. In one preferred embodiment of this invention,monolithic polymer rods with size of 50 mm×10 mm I.D. are used asfillers. In another preferred embodiment of this invention, monolithicpolymer rods with size of 10 mm×34 mm I.D. are used as fillers. Inanother preferred embodiment of this invention, the monolithic polymercylinder with various inner and outer diameters are used as fillers.

The performance of capillary columns can be improved by our inventionsdescribed earlier. One method is to choose right combination ofporogenic solvents to generate separation media with and withoutpressure. The choice of solvents with right polarity and solventingpower of the polymers can result in porous polymer support with nomicropores or small pores which can affect separation efficiency of thecolumn. Exertion of pressure during polymerization will further improvethe uniformity of the media and avoid the formation of micropores. Inone embodiment, the capillary columns of internal diameter 320 μm isprepared with the combination of solvents including chlorocyclohexaneand 1-decanol to generate the monolithic porous polymer supportcontaining no micropores or small pores which result in poor masstransfer of the sample molecules with and without the pressure of 120psi. In another embodiment, the combination of solvents including1-ethylhexanoic acid and mineral oil has been used to prepare monolithicporous materials containing no micropores with and without pressure.

The employment of x-ray, UV-vis can improve the performance of capillarygreatly. X-ray can penetrate materials low energy and intensity loss.X-ray can penetrate a capillary with almost no intensity loss.Sensitizers or scintillators can absorb x-ray energy transferred by thesolvents effectively and emit fluorescence or phosphorescence lighthomogeneously in the solutions. The homogeneous fluorescence andphosphorescence light can be absorbed by initiators which initiate thepolymerization homogeneously in the polymerization solutions. This leadsto homogeneity of the porous structure of the porous polymer supportincluding monolithic separation media and particles. This improves thecolumn efficiency of the monolithic capillary greatly. The lowtemperature polymerization using x-ray as energy source results in slowpolymerization rate due to the lower polymerization temperature. Finetuning the intensity and energy of x-ray results in the desiredpolymerization rate which can lead to the formation of homogeneousseparation media. The empolyment of x-ray, scintillators/sensitizers,solvents with right solventing powers and the pressure duringpolymerization can leads to the formation of separation media with nomicropores or small pores with similar size to the sample moleculeswhich leads to greatly improved performance. In one embodiment,capillary columns have been prepared using x-ray as energy source. Inanother embodiment, microchip columns have been prepared with x-ray asenergy source.

The right choice of solvents with reflective index very close to thepolymers allows the light to travel through the capillary with littleloss of intensity of the light, which is known as Christiansen Effectand described earlier. The use of initiators having Bleaching Effectallows the UV-vis light to travel through the capillary columns withnegligible loss of intensity. The lights are homogeneous in thepolymerization solutions. The absorptions of the homogeneous lightresult in homogeneous initiations of the polymerization which is unlikethe initiation promoted by thermal heating or the UV-vis initiatedpolymerization without the consideration Christiansen and bleachingeffect. This leads to the formation of homogeneous polymerization in thesolution. As the result, more homogeneous porous structure can beformed. This leads to the much improved performance of the capillarycolumns. The combination of the above method with solvents of goodsolvating power, pressurized polymerization can lead to formation of thehomogeneous media containing no micropores or small pores with having noimpact on column performance. In one embodiment, capillary columns havebeen prepared using UV-visible light. In another embodiment, microchipcolumns have been prepared using UV-vis light.

EXAMPLES

While many other values of the variables in the following examples canbe selected from this description with predictable results, thefollowing non-limiting examples illustrate the inventions:

General

In general, the preparation of each type of media in the followingexamples include three major steps, which are:(1) preparation of polymermatrix; (2) modification of the polymer matrix to contain desiredfunctional groups; and (3) characterization of the media.

Firstly, the preparation major step, includes several substeps, whichare: (1a) formulation of the polymerization mixture by varying the typesand amount of monomers, porogens and initiators; (1b) degassing thepolymerization mixture by vacuum and helium purge. (1c) assembly of theempty column with different diameter and material of tubing as a mold,frequently with one end of the column sealed with a cap or stopper; (1d)filling the column with the polymerization mixture; (1e) sealing theother end of the mold with a cap or a specially designed fitting to addpressure during the polymerization; (1f) preheating the solution in themold with one open end if a glass column is used and applying a selectedpressure using various pressure sources including hydraulic pressure,air pressure or mechanic pressure; (1g) placing the mold in atemperature-controlled heating bath or oven at a selected temperature;(1h) polymerizing for various amount of time; (1i) taking the column outof the heating bath after polymerization and replacing the sealing capor pressurization device with column fittings for pumping the washingliquid through; (1j) washing the column with organic solvents and/orwater.

Secondly, the modification process includes: (2a) formulating themodification reaction mixture with various types and amount of reactantsand catalysts; (2b) pumping more than 5 bed volume modification solutionthrough the column and sealing it, or pumping more solutioncontinuously. (2c) carrying out the modification reaction at varioustemperature and time in a temperature-controlled heating bath; (2d)washing the column with organic and water.

The columns are characterized using varieties of methods includingliquid chromatography separation, porosimetry, BET surface areameasurement, Scanning Electron Microscopy, UV spectroscopy and visualobservation. Liquid chromatography characterization includes variousmodes of separation at different speeds. The commonly used devices,processes and methods are described in the following preceding thespecific examples.

Degassing of the Polymerization Solution

The polymerization mixture is degassed by vacuum generated by wateraspirator for 5 minutes using an ultrasonic degasser. It is followed bypurging the solution for minimum of 20 minutes.

Stabilizing and Conditioning Methods

The ion exchange columns were subject to a stabilizing and conditioningprocedure following the washing step after modification reaction. Thestabilizing and conditioning procedure for a glass column (100×10 mmI.D.) of strong anion exchanger was as following: The flow rate of 0.01mol/I Tris.HCl buffer at pH 7.6 was increased linearly from 0 ml/min to20 ml/min in 1 minute, and kept for 0.5 minutes. The compression liquidwas changed to 1 mol/I NaCl in the same buffer by gradient in 2 minutes,kept for 0.5 minutes. The stabilizing and conditioning procedure for aPEEK-lined stainless steel column is the same as above except themaximum flow rate was 5 ml/min instead of 20 ml/min. The procedures forother ion exchange columns depend on the maximum flow rate allowed.

Characterization Procedures

1. Characterizations with Liquid Chromatography (LC) Separations

1a. LC Characterization Method 1: Liquid Chromatography Separation ofProteins and Peptides

Mobile phases:

-   -   Mobile phase A (or Buffer A):    -   Anion Exchange Chromatography: 0.01 M Tris.HCl (pH 7.6)    -   Cation Exchange Chromatography: 0.01 M sodium phosphate (pH 7.0)    -   Reversed Phase Chromatography: 0.15% Trifluoroacetic acid (TFA)        in water

Mobile phase B (or Buffer B):

Ion Exchange Chromatography: 1 M NaCl in Buffer A

Reversed Phase Chromatography: 0.15% TFA in acetonitrile (ACN)chromatography Samples:

Anion Exchange Chromatography:

0.6 mg/ml myoglobin, 1 mg/ml conalbumin, 1 mg/ml ovalbumin and 1 mg/mltrypsin inhibitor.

Cation Exchange Chromatography:

1 mg/ml connalbumin, 1 mg/ml ovalbumin and 1 mg/ml trypsin inhibitor.

Reversed Phase Chromatography for proteins:

1.5 mg/ml Ribonuclease A, 0.5 mg/ml Cytochrome C, 1.5 mg/ml BSA, 0.9mg/ml

Carbonic Anhydrase, 1.5 mg/ml Ovalbumin.

Reversed phase chromatography for peptides:

33 ?g/ml Met-Enkephalin, Let-Enkephalin, Angiotensin II, Physalaemin,Substance P

Sample Preparation:

Filled 8 ml of buffer A in a 15 ml graduated plastic sample tube;Weighed appropriate amount of protein samples and placed them into thissample tube; Sealed the tube with cap and tumbled the tube gently untilall the proteins dissolved; Added in more buffer solution until the 10ml mark on the sample tube was reached.

A column was characterized by protein separation according to thefollowing procedure: The column was attached to an Isco 2350 Two PumpSystem. Pump A contained 0.01 mol/l Tris.HCl buffer (Buffer A) and PumpB contained 1 mol/l NaCl in Buffer A (Buffer B). The mobile phases weredegassed with helium purging for more than 20 minutes before use. The UVdetector was set at 0.05 sensitivity and 280 nm wavelength for proteinseparation (214 nm for peptide separation). The volume of the sampleinjection was 20 micro liters. The column was first cleaned by 20 bedvolume of Buffer B and conditioned by 15 bed volume of Buffer A at the 3m/min for 4.6 mm I.D. column (10 ml/min for 10 mm I.D. column). Theseparation was achieved by a gradient from 0 to 50% Buffer B for 20 bedvolume at the flow rate of 3 ml/min for 4.6 mm I.D. column and 10 ml/minfor 10 mm I.D. column.

1b. LC Characterization Method 2: Binding Capacity Measurement of IonExchangers

The binding capacity of an ion exchange column was measured by frontalanalysis. The column was cleaned with 20 bed volume of Buffer B andconditioned with 15 bed volume of Buffer A. This columns was saturatedwith the sample protein by pumping 5 mg/ml BSA or lysozyme solution (BSAfor anion exchanger and reversed phase, and lysozyme for cationexchanger) in Buffer A through the column until no further increase ofthe absorbance of the eluent, followed by cleaning the non-adsorbedproteins with 100% Buffer A. The protein bound to the columns was elutedby a gradient from 0 to 50% Buffer B for 20 bed volume. The elutedprotein was collected in a sample vial and the protein concentration wasdetermined by UV spectrometer at 280 nm. The total binding capacity ofthe column was calculated by multiplying the concentration of collectedprotein with the volume of collection.

1c. LC Characterization Method 3: Hydrophobic Interaction Chromatography

This column was characterized for hydrophobic interaction chromatographyof proteins. A mixture of proteins containing Ribonuclease, CytochromeC, Lysozyme, Bovine Serum Albumin and Carbonic Anhydrase (1, 0.3, 0.2, 1and 0.5 mg/ml in 0.01 M Tris.HCl buffer solution at pH 7.0.) wasseparated by a 15 minute gradient of 0.5 moldi NaCl in 0.01 mol/ITris.HCl buffer (pH 7.6) to the same buffer at the flow rate of 1ml/min.

1d. LC Characterization Method 4: Polymer Molecular Weight Determinationby Precipitation-Redissolution Chromatography

Characterization method 6 was used for polymer molecular weightdetermination using Precipitation/Redissolution Chromatography. Sevenpolymerstandards(Mp: 12,900, 20,650, 34,500, 50,400, 96,000, 214,500,982,000) were separated by a 6 minute gradient from 15% to 80% THF inmethanol at the flow rate of 2.6 ml/min. The polymer standards weredissolved in 50% THF in methanol with the total concentration of 56mg/ml. The injection volume was 20 Fl.

1e. LC Separation of Nucleotides

A sample of Pd(A)₁₂₋₁₈ (2.4 mg/ml in water) was separated by a gradientfrom 30% to 60% Buffer B in Buffer A (A=20% acetonitrile and 80% 20mmol/I sodium phosphate; B=1 mol/I NaCl in A) at the flow rate of 1ml/min.

1f. LC Separation of Nucleotides with Anion Exchanger

A sample of AMP, ADP and ATP (0.4, 0.8, 0.8 mg/ml in water respectively)was separated in a gradient of 0 to 50% Buffer B in Buffer A asspecified in LC Characterization Method 1.

Numbered Examples Example 1

A polymerization solution was prepared as following: Weighed 1.2 g ofglycidyl methacrylate (GMA), 0.80 g of ethylene dimethacrylate (EDMA)(polymerization mixture 1) and 0.02 g of 2,2=-azobisisobutyronitrile(AIBN) into a 20 ml sample vial and shook the mixture gently until itbecame a homogeneous solution; Weighed 2.55 g of cyclohexanol (CHOH) and0.45 g of dodecanol (DODOH) into this solution and shook it until it ishomogeneous. The polymerization mixture was degassed as in DegassingProcedure.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),one end of which was sealed by a pressuring device shown in FIG. 4, wasfilled with this solution until the column is full. A PEEK-plugcontained in the stainless steel screw cap, which was the original capfor the column shown in the same figure, was used to seal the other endof the column. The device of FIG. 3 was connected to a syringe pump.Water was used as the medium to generate the pressure of 120 psi. No airwas inside the column. This column was placed into a water bath uprightat 60?C and kept for 20 hours. After polymerization, the column wastaken out of the water bath and cooled to room temperature.

The device of FIG. 3 was detached from the syringe pump after thepressure was released. The device of FIG. 3 was opened and carefullyremoved from the column. White polymer extended outside the column. Thelength of the polymer was found to be about 2 mm shorter than the heightof the polymerization solution inside the column and the device of FIG.3. This extended part of polymer was removed by razor blade. The columnwas then fitted with the original HPLC column fittings. The column wasconnected to a HPLC pump and washed with acetonitrile at 0.5 ml/min for20 minutes at 45?C.

The fillings from one end of the column were detached and the media waspressed out of the column by pumping 10 m/min acetonitrile into thecolumn through the other end. The wall surface of the polymer media wasfound to be smooth. The top of polymer was flat.

Comparative Versions of Example 1

A polymerization solution was prepared as following: Weighed 1.2 g ofglycidyl methacrylate (GMA), 0.80 g of ethylene dimethacrylate (EDMA)(polymerization mixture 1) and 0.02 g of 2,2=-azobisisobutyronitrile(AIBN) into a 20 ml sample vial and shook the mixture gently until itbecame a homogeneous solution; Weighed 2.55 g of cyclohexanol (CHOH) and0.45 g of dodecanol (DODOH) into this solution and shook it until it ishomogeneous. The polymerization solution was degassed with the aboveDegassing Procedure.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),one end of which was sealed by a PEEK-plug contained in the stainlesssteel screw cap which was the original cap for the column. This columnwas filled with the above solution until it was full. A PEEK-plug wascarefully placed on the top of column and sealed with another screw cap.No air should be kept inside the column. This column was placed into awater bath upright at 60?C and kept for 20 hours. After polymerization,the column was taken out of the water bath and cooled to roomtemperature.

The PEEK-plugs were detached and white polymer was observed in thecolumn. The column was then fitted with the original HPLC columnfittings. The column was connected to a HPLC pump and washed withtetrahydrofuran (THF) at 0.5 ml/min for 20 minutes.

The fitting from one end of the column was detached and the media waspressed out of the column by pumping 10 ml/min acetonitrile (ACN) intothe column through the other end. The wall surface of the media wasfound to contain many small irregular dents.

Other columns were prepared using different monomers. Irregular voidswere found on the wall surface of the polymer rods. Pictures of two ofthese rods and one rod made under pressure were taken and shown in theFIG. 7.

Alternative Versions of Example 1

The procedure of Example 1 was followed except that differentpolymerization mixtures were used having different proportions andcombinations of the functional monomers and crosslinkers. The functionalmonomers used includes glycidyl methacrylate (GMA), 2-hydroethylmethacrylate (HEMA), methyl methacrylate (MMA),2-(acryloyloxyethyl)trimethylammonium methyl sulfate (ATMS), acrylicacid (AA), 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS), stearylmethacrylate (SMA), lauryl methacrylate (LMA), butyl methacrylate (BMA),styrene (ST) and 4-ethylstyrene (EST). The crosslinking monomers(crosslinkers) used include ethyleneglycol dimethacrylate (EDMA),divinyle benzene (DVB). Different proportions of functional monomers,crosslinking monomers and porogens were used. The porogens includesdifferent alcohols such as cyclohexanol, dodecanol, decanol,1-hexadecanol, butanol, propanol, iso-propanol, ethanol, methanol,1,4-butanediol and others such as toluene, N,N-Dimethyl acetamide,acetonitrile, 1,2-dimethoxyethane, 1,2-dichloroethane, dimethylphthalate, 2,2,4-trimethylpentane, 1,4-dixane, 2-methyloxyethanol,1,4-butanediol, m-xylene, diisobutyl phthalate, tetra(ethylene glycol)dimethyl ether, tetra(ethylene glycol), poly(propylene glycol) (F.W.1000), poly(propylene glycol) monobutyl ether (F.W. 340, 1000, 2500).The initiators used included 2,2-prime-azobisobutyronitrile (AIBN) andbenzoyl peroxide.

For each of the combinations of monomers and porogens, columns wereprepared, examined and characterized.

Several columns were made with each polymerization mixtures underseveral pressure conditions. The pressure conditions include: (1) thecolumn opened to the atmosphere during polymerization; (2) the columnsealed during polymerization; (3) pressure being applied to the columnwith gas applied directly to the polymerization mixture using nitrogenas the gas; (4) each of rubber, plastic and metal pistons being incontact with the polymerization mixture and applying pressure fromeither a spring, hydraulic pressure, gas pressure or by threading thepiston downwardly using the device described above or the modifieddevice when the mechanical force such as spring was used.

The results were: (1) for cases when atmospheric pressure was presentthere were discontinuities on the surface of a high percentage ofcolumns. Column efficiencies and resolutions were not reproducible; (2)for pressure conditions in which the column was sealed, a higherpercentage of columns had discontinuities on the surface, columnefficiencies and resolutions were not reproducible; (3) for columns inwhich gas pressure was applied, the top end surfaces of the columns weresoft and irregular, and the wall surfaces were smooth. The separationchromatograms had better reproducibility. The elution peaks are sharperthan when pressure was not applied; (4) reproducibility was very highwhen pistons were used and the resolution was better than all the abovemethods used.

Example 2

A polymerization solution was prepared as Example 1 with the followingpolymerization solution: 2.0 g GMA, 2.5 g2-(acryloyloxyethyl)trimethylammonium methyl sulfate (ATMS, 80%), 6.0 gEDMA, 7.5 g 1,4-butariediol, 6.75 g propanol, 0.75 g water and 0.1 gAIBN.

An empty glass column (10 mm inner i.d. and 100 mm length), one end ofwhich was sealed by a pressuring device shown in FIG. 4, was filled withthis solution until the column is full. A TEFLON-plug contained in thePEEK screw cap shown in the same figure, was used to seal the other endof the column. The device of FIG. 4 was connected to a syringe pump. Noair was inside the column. This column was placed into a water bathupright at 60?C and kept for 15 minutes. Then the column was pressurizedto 120 psi by syringe pump using water as the medium, and kept for 20hours. After polymerization, the column was taken out of the water bathand cooled to room temperature.

The device of FIG. 4 was detached from the syringe pump after thepressure was released. The device of FIG. 4 was opened and carefullyremoved from the column. It was found that the height of the polymer rodwas 4 mm shorter than the height of the polymerization solution insidethe column. The column was then fitted with the original HPLC columnfittings. The column was connected to a HPLC pump and washed withacetonitrile at 2 ml/min for 20 minutes at 45?C.

The fittings from one end of the column were detached and the media waspressed out of the column by pumping 10 ml/min acetonitrile into thecolumn through the other end. The wall surface of the polymer media wasfound to be smooth. The top of polymer was flat.

Alternative Versions of Example 2

The method of Example 2 was followed with the change of pressures andmethods of applying the pressures. Different constant pressures wereused during polymerization. The pressures used include 80 psi, 150 psi,180 psi, 200 psi, 240 psi and 300 psi. The back pressures of thesecolumns are different. The Scanning Electron Microscopy examination ofthe polymer structure revealed that the particle sizes of these polymersare also different.

A step gradient of pressure was applied to the polymerization mixtureduring polymerization. The gradient is as following: 4 psi/min increasefrom 10 psi for 5 min, 2 psi/min increase for 10 min, 1 psi/min increasefor 20 min, 0.8 psi/min increase for 30 min and then increase thepressure to 180 psi within an hour. The final pressure of 180 psi waskept for 20 hours during polymerization.

A linear gradient from 15 psi to 180 psi for 2 hours was used topressurize the reaction at early stage of polymerization. 180 psi waskept for another 18 hours during the rest of polymerization.

All these columns were modified by pumping in 5 ml solution of 0.45 g/mltrimethylamine hydrochloride in trimethylamine aqueous solution (50%volume). The columns were sealed and heated in a water bath at 40° C.for 3 hours. They were washed with 20 bed volume of water right aftermodification. These columns were subjected to Bed Stabilization andConditioning as described above. They were characterized as above LCCharacterization Method for ion exchangers. The back pressures, particlesizes and separation resolutions of these columns all varied with thepressurization methods.

Different polymerization time was also used. A column was prepared as inexample 2 except that the polymerization time was 44 hours instead of 20hours.

Ten columns were prepared in parallel using a ten channel manifold withonly one pressure source. The columns prepared have betterreproducibility than individual preparation process.

The polymer morphologies and the internal structures of the particleswere examined with Scanning Electron Microscopy (SEM). It was found thatthe internal structures of these particles are non-porous instead ofporous in the other monolithic media known in the art.

Example 3

Two columns were prepared as in Example 1 and 2 with the followingsolution: 17.5 g DVB, 19.8 g tetra(ethylene glycol), 10.2 gtetra(ethylene glycol) dimethyl ether, and 0.18 g AIBN. After wash withacetonitrile, the column was further washed with 20 bed volume of waterat the flow rate of 16 ml/min. The manually positioned pistons in bothends were compressed into the column. This column was then washed withacetonitrile containing 0.15% trifluoroacetic acid and characterized byLC characterization method la and lb. The resolutions of these columnsfor both protein and peptide separation were improved greatly over thecolumns made in the Comparative Examples. The capacities were more than5 times higher. The back pressures of these columns were low. Highresolutions were achieved with the mobile phase gradient starting from100% water containing 0.15% TFA. No wall effect was found in thesecolumns. The proteins and peptides pre-eluted out of the column in thecolumns from Comparative Examples due to wall effect in aqueous phase.

Comparative Versions of Example 3

A polymerization solution was prepared as Comparative Example of Example1 with the following reagents: 3 ml styrene, 2 ml divinylbenzene, 7.5 mldodecanol and 0.5 g AIBN. The column was characterized withreversed-phase protein and peptide separation described as LCCharacterization 1a and 1b.

Alternative Versions of Example 3

Example 3 was followed with different combinations of porogens,different monomers containing different carbon chain length, differentamount of total monomer contents, different initiators and differentshrinkage solvents.

The porogens used includes: alcohols containing C1 to C12, N,N-Dimethylacetamide, acetonitrile, 1,2-dimethoxyethane, 1,2-dichloroethane,dimethyl phthalate, 2,2,4-trimethylpentane, 1,4-dixane,2-methyloxyethanol, 1,4-butanediol, toluene, m-xylene, diisobutylphthalate, tetra(ethylene glycol) dimethyl ether, tetra(ethyleneglycol), poly(propylene glycol) (F.W. 1000), poly(propylene glycol)monobutyl ether (F.W. 340, 1000, 2500). The combination of some of thesesolvents led to high resolution columns as well. The alcohols and theircombinations can provide large channels for mobile phase to flow throughwith low back pressure while providing high resolutions. The resolutioncan be finely tuned with other good solvents as well.

The monomers used include butyl methacrylate and stearyl methacrylatewith the above combination of porogens. One column was prepared with thefollowing polymerization solution: 7 g SMA, 10.5 g DVB, 19.5 g ethanol,13.0 g butanol and 0.18 g AIBN. Another column was prepared with thefollowing polymerization solution: 7 g lauryl methacrylate (LMA), 1 gHEMA, 12 g EDMA, 30 g dodecanol and 0.2 g AIBN. Another column wasprepared with the following polymerization solution: 7 g butylmethacrylate (BMA), 1 g HEMA, 12 g EDMA, 3 g water, 16.5 g propanol,10.5 g 1,4-butanediol and 0.2 g AIBN. The combinations of these monomerscontaining different carbon chain length provide differenthydrophobicity and interaction, which offer high resolution and recoverytoward samples with different hydrophobicity and characteristics. Forexample, the butyl methacrylate based media was used for morehydrophobic protein separation and the stearyl methacrylate based mediacan be used for more hydrophilic protein, peptide or oligonuleotideseparations.

Different ratios of monomer to crosslinker were also used to tune theselectivity and resolution. One column was prepared with the followingpolymerization solution: 1.05 g SMA, 0.7 g DVB, 3.25 g ethanol and 0.018g AIBN.

Different initiator was also used. A column was prepared with thefollowing polymerization solution: 10 ml divinylbenzene (80% purity), 30ml dodecanol, 10 ml styrene and 0.20 g benzoyl peroxide.

Different polymerization time was also used. A column was prepared as inexample 3 except that the polymerization time was 44 hours instead of 20hours.

Different polar solvents were used to shrink the polymer beforecompression. A column was prepared as Example 3 and washed with 20 bedvolume of 1 M NaCl after water wash. Manually positioned pistons werecompressed into the column after the salt wash. The column show no walleffect when 0.1 M NaH₂PO₄ (pH 4.0) was used as the starting mobilephase.

These experiments were repeated with different washing solutions anddifferent catalysts with and without pressure in the presence of anaqueous solution after the plug was polymerized as well as withdifferent pressures. In each case, when no pressure was applied, therewere discontinuities in the outer wall, and upon characterization, therewas a lack of repeatability and the peaks of the chromatograms were lesspronounced when no pressure was applied after swelling by washing withan aqueous solution.

Tests have been run at a plurality of pressures both low pressures andhigh pressures including 60 psi (pounds per square inch) and 120 psi and600 psi with good results. It is believed that the amount of pressureneeded will vary with the diameter of the column and the particularpolymerization mixture but satisfactory results can be obtained at avery low pressure in all cases. The upper limit on pressure is thestrength of the column walls and fittings.

The amount of pressure also affects the pore size so that the pressureshould be selected together with desired pore size, distribution andreproducibility of the column.

Example 4

A column was prepared as Example 2 with the following solution: Nine gof glycidyl methacrylate, 9 g of ethylene dimethacrylate, 0.18 g, 21.6 gof cyclohexanol and 6.3 g of dodecanol. The length of the polymer wasfound to be about 7 mm shorter than the height of the polymerizationsolution inside the column. The column was then fitted with the originalcolumn fittings. The column was connected to a HPLC pump and washed withacetonitrile at 4 ml/min for 20 minutes at 45?C. The column was furthermodified as following:

A solution containing 570 mg trimethylamine hydrochloride, 24 mldiethylamine and 6 ml water was pumped into these columns at the flowrate of 2 ml/min for 18 minutes. These columns were then placed in awater bath at 30?C for 3 hour. Each column was washed with 100 ml water.The columns were further washed with 0.01 mol Tris.HCl buffer at pH 7.6at 4 ml/min for 30 minutes. The column was stabilized and conditioned bySTABILIZING AND CONDITIONING METHODS. The pistons from original columnfittings were compressed in completely after the wash. The column backpressures were about 360 psi at 10 ml/min in this buffer. The column wascharacterized as LC Characterization Method described above.

Example 5

A polymerization solution was prepared by mixing 1 g styrene, 1 gdivinylbenzene (DVB) (80% divinyl benzene and 20% ethylstyrene (EST)), 3g dodecanol and 0.02 g AIBN.

This solution was degassed by N₂ purging for 20 minutes and filled intoa stainless steel column (50×4.6 mm i.d.), one end of which was sealedwith a PEEK plug inside the screw cap from the column fittings. Theother end of the column was sealed with another PEEK plug. It waspolymerized at 70 degrees C. in a water bath for 24 hours. This columnwas fitted with the original column fittings and washed with THF at theflow rate of 1 ml/min for 10 minutes before it was used for separationof proteins. The back pressure of this column was about 230 psi at theflow rate of 10 ml/min. The compression of the polymer at 10 ml/min ofacetonitrile was about 2.9 mm. This column was used for reversed phaseprotein and peptide separation as LC Characterization Method 1a and 1b.

Alternative Versions of Example 5

Another column was prepared as in example 5 but with higher totalmonomer contents. The polymerization solution contained 1.2 g styrene,1.2 g divinylbenzene, and 2.6 g dodecanol and 0.024 g AIBN. The backpressure of the column was 220 psi at 10 ml/min acetonitrile and thecompression of the polymer was only 0.9 mm. The higher total monomercontent makes the column less compressible.

Columns of different diameters including 22 mm, 1 5 mm, 10 mm, 8 mm, 2.1mm, 1 mm, 542 ?m, and 320 ?m were prepared as in Example 4. Shortercolumns with the size of 10 mm×2.1 mm i.d. were also prepared. Thesecolumns were characterized with reversed phase protein separation by LCCharacterization Method but at the same flow velocity corresponding tothe diameters of the columns. The in situ polymerization method isapplicable in columns with different diameters. It is especially usefulfor smaller diameter columns or micro fluid channels since there is noother packing step involved.

Example 6

Another column was prepared according to Example 5.

This column was fitted with the original column fittings containing apiston and washed with acetonitrile at the flow rate of 1 ml/min for 10minutes. The column was further washed with water for 10 minutes. Thepistons in both ends were compressed into the column. This column wascharacterized by LC Characterization Method. The compression of thepolymer in water by piston and held by this piston after compressionavoid the wall effect resulted from the shrinkage of polymer in water.

Alternative Versions of Example 6

Another column was prepared as Example 5 with the following solution:1.8 g divinylbenzene, 0.2 g of styrene, 2.3375 g dodecanol, 0.6625 gtoulene, 0.02 g of AIBN and 3.0 g of dodecanol. All the reagents weredegassed by vacuum using aspirator for five minutes, followed by purgingwith Helium for 20 minutes, before weighing. The polymerization solutionwas filled into a stainless steel column (50×4.6 mm i.d.), one end ofwhich was sealed by PEEK-plug contained in the screw cap. The other endof the column was sealed with another PEEK plug. It was polymerized at66 degrees C. in a water bath for 24 hours. This column was fitted withthe original column fittings containing a piston and washed withacetonitrile at the flow rate of 1 ml/min for 10 minutes. The column wasfurther washed with water for 10 minutes. The pistons in both ends werecompressed into the column. This column was characterized by the LCCharacterization Method. The higher content of crosslinker improved thecompacity. This version of a column has more than 3 times highercapacity than the some other columns prepared in accordance with Example6.

Another column was prepared with the following polymerization solution:10 ml divinylbenzene (80% purity), 10 ml styrene and 0.20 g benzoylperoxide.

This polymerization solution was purged by N₂ for 20 minutes. It wasfilled into a stainless steel column (50×4.6 mm i.d.), one end of whichwas sealed by PEEK-plug contained in the screw cap. The other end of thecolumn was sealed with another PEEK plug. It was polymerized at 70?C ina water bath for 24 hours. This column was fitted with the originalcolumn fittings and washed with tetrahydrofuran at the flow rate of 1ml/min for 10 minutes. It was characterized by reversed phase proteinand peptide separation as described in LC Characterization Method.Different initiators such as benzoyl is also effective in making themonolithic media.

A stainless column of smaller size (50×2.1 mm i.d.) and a PEEK column(50×4.6 mm i.d.) were prepared and characterized 8 as above example.

Example 7

A column was prepared as in Example 6 with the following solution:divinylbenzene, 0.2 g of hydroxylethylmethacrylate and 0.02 g of AIBN.It was polymerized at 70?C in a water bath for 24 hours. This column wasfitted with the original column fittings containing pistons. It waswashed with acetonitrile at the flow rate of 1 ml/min for 10 minutes,and further washed with water and 0.5 mol/I NaClin 0.01 mol/I Tris.HC1buffer (pH 7.6). This column was characterized by reversed-phase proteinseparation as in LC Characterization Method.

Alternative Versions of Example 7

A column was prepared and characterized according to the above procedureexcept the weight of hydroxylethylmethacrylate and divinylbenzene werechanged to 0.4 g and 1.6 g.

Another column was prepared and characterized according to the aboveprocedure except the weight of hydroxylethylmethacrylate anddivinylbenzene were changed to 1 g and 1 g.

Another column was prepared as in Example 7 with the followingpolymerization solution: −1.8 g divinylbenzene, 0.16 g styrene, 0.04 gof hydroxylethylmethacrylate, and 0.02 g of AIBN and

This column was fitted with the original column fittings containingpistons after polymerization. It was washed with acetonitrile at theflow rate of 1 ml/min for 10 minutes, and further washed with water and0.5 mol/l NaClin 0.01 mol/l Tris.HCl buffer (pH 7.6). This column wascompressed with pistons and characterized by reversed phase separationsof proteins and peptides as in LC Characterization Method.

Example 8

An empty syringe barrel (70×12 mm i.d. Redisep barrel for Combiflashchromatography from Isco, Inc., 4700 Superior Street, Lincoln, Nebr.68504) was sealed at one end and filled with the followingpolymerization solution: 1.6 g hydroxylethyl methacrylate, 6.4 gdivinylbenzene, 89 mg AIBN, 12 g dodecanol after degassing with N₂ for20 minutes. The tip of the barrel was sealed with a blocked needle. Thisbarrel was heated in a water bath at 70?C for 24 hours. It was connectedto a HPLC pump and washed with THF at the flow rate of 1 ml/min for 30minutes. It was then used for both normal phase and reversed phaseseparation of phenolic compounds.

Example 9

A polymerization solution was prepared as following: Weighed 1 g ofhydroxylethyl methacrylate, 1 g of ethylene dimethacrylate and 0.02 g ofAIBN into a 20 ml sample vial and shook the mixture gently until itbecame a homogeneous solution; Weighed 1 g of cyclohexanol and 2 g ofdodecanol into this solution and shook it until it is homogeneous. Allthe reagents were degassed by vacuum using aspirator for five minutes,followed by purging with Helium for 20 minutes, before weighing.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),one end of which was sealed by a device of FIG. 4 was filled with thissolution until the column is full. A PEEK-plug contained in thestainless steel screw cap, which was the original cap for the column,was used to seal the other end of the column. The device of FIG. 4 wasconnected to a syringe pump. Water was used as the medium to generatethe pressure of 120 psi. No air should be kept inside the column. Thiscolumn was placed into a water bath upright at 60?C and kept for 20hours. After polymerization, the column was taken out of the water bathand cooled to room temperature. The column was connected to a HPLC pumpand washed with dry THF (dried by molecular sieve) at 0.5 ml/min for 20minutes. The column was used for Normal Phase separation of drugs.

Alternative Versions of Example 9

A column was prepared as in Example 9 with the following polymerizationsolution: 0.5 g GMA, 0.5 g HEMA, 1 g EDMA, 1.8 g cyclohexanol, 1.2 gdodecanol, 0.02 g AIBN.

The columns were washed with water after THF wash at the flow rate of0.5 ml/min for 20 minutes. 10 ml of 1.0 mol/I sulfuric acid in water waspumped through the columns. The columns were sealed with column plugsand placed into a water bath at 80?C for 3 hours. They were washed with20 ml water after the modification reaction and further washed with dryTHF before characterization with Normal Phase separation.

Another column was prepared with the following polymerization solution:1 g glycidyl methacrylate, 1 g ethylene dimethacrylate, 2.4 gcyclohexanol, 0.6 g dodecanol, 0.02 g AIBN.

The columns were washed with water after THF wash at the flow rate of0.5 ml/min for 20 minutes. 10 ml of 1.0 mol/l sulfuric acid in water waspumped through the columns. The columns were sealed with column plugsand placed into a water bath at 80?C for 3 hours. They were washed with20 ml water after the modification reaction and further washed with dryTHF before characterization with Normal Phase separation.

Example 10

A stainless steel column (50×4.6 mm i.d.) was prepared as in Example 3with the following polymerization solution contained 0.7 g laurylmethacrylate (LMA), 0.1 g HEMA, 1.2 g EDMA, 3 g dodecanol and 0.02 gAIBN.

Alternative Versions of Example 10

A column was prepared with the following polymerization solutioncontained 0.8 g lauryl methacrylate (LMA), 1.2 g EDMA, 3 g dodecanol and0.02 g AIBN. Another column was prepared with the followingpolymerization solution containing 0.175 g stearyl methacrylate (SMA),1.575 g DVB (80% pure), 3.25 g 1-hexadecanol and 0.018 mg AIBN to form amixture with a ratio SMA/DVB=10/90. Another column was prepared with thefollowing polymerization solution containing 0.7 g SMA, 1.05 g DVB, 3.25g octanol and 0.018 g AIBN to provide a ratio of 40/60 SMA/DVB.

Another column was prepared with the following polymerization solutioncontaining 1.05 g SMA, 0.7 g DVB, 3.25 g ethanol and 0.018 g AIBN toprovide a ratio of 60/40 SMA/DVB.

Another column was prepared with the following polymerization solutioncontaining 0.7 SMA, 1.05 g DVB, 1.95 g ethanol, 1.30 g butanol and 0.018g AIBN to provide a ratio of 40/60 SMA/DVB.

Other columns were prepared as above using tetradecanol, decanol,octanol, hexanol, butanol, propanol, ethanol and methanol and theircombinations as porogenic solvents.

Another column was prepared as above with the following polymerizationsolution: 0.7 SMA, 1.05 g DVB, 2.925 g ethanol, 0.33 g methanol, 0.33 gisopropanol and 0.018 g AIBN to provide ratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerizationsolution: 0.7 SMA, 1.05 g DVB, 2.6 g ethanol, 0.33 g methanol, 0.33 gpropanol, 0.33 g butanol and 0.018 g AIBN to provide a ratio of 40/60SMA/DVB.

Another column was prepared as above with the following polymerizationsolution: 0.7 SMA, 1.05 g DVB, 2.762 g ethanol, 0.33 g methanol, 0.33 gpropanol, 0.33 g butanol, 0.33 g hexanol and 0.018 g AIBN to provide aratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerizationsolution: 0.7 SMA, 1.05 g DVB, 2.435 g ethanol, 0.33 g methanol, 0.33 gpropanol, 0.33 g butanol, 0.33 g hexanol, 0.33 g octanol and 0.01 8 gAIBN to provide a ratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerizationsolution: 1.05 g DVB, 3.08 g ethanol, 0.0.16 g ethyl ester and 0.018 gAIBN. This column was used for peptide separation as in Example 10, toprovide a ratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerizationsolution: 1.75 g DVB, 3.25 g dodecanol and 0.018 g AIBN. This column wasused for peptide separation as in the first version of example 10. Aseries of columns with alcohols containing C1 to C12 were prepared asabove and characterized with peptide separation.

A series of columns were prepared as above using the following porogensinstead of dodecanol: iso-propanol, N,N-Dimethyl acetamide,acetonitrile, 1,2-dimethoxyethane, 1,2-dichloroethane, dimethylphthalate, 2,2,4-trimethylpentane, 1,4-dixane, 2-methyloxyethanol,1,4-butanediol, toluene, m-xylene, diisobutyl phthalate, tetra(ethyleneglycol) dimethyl ether, tetra(ethylene glycol), poly(propylene glycol)(F.W. 1000), poly(propylene glycol) monobutyl ether (F.W. 340, 1000,2500).

Another column was prepared as above with the following polymerizationsolution: 1.75 g DVB, 2.925 g isopropanol, 0.325 g 1,4-butanediol and0.018 g AIBN. Another column was prepared as above with the followingpolymerization solution: 1.75 g DVB, 2.275 g isopropanol, 0.975 g2-methyloxyethanol and 0.018 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 1.75 g DVB, 2.60 g isopropanol, 0.65 dimethyl phthalate and0.018 g AIBN. Another column was prepared as above with the followingpolymerization solution: 1.75 g DVB, 2.7 g tetraethylene glycol, 0.3 gdiethylene glycol and 0.018 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 1.75 g DVB, 2.7 g tetra(ethylene glycol), 0.3 g glycerol and0.018 g AIBN. Another column was prepared as above with the followingpolymerization solution: 1.75 g DVB, 1.98 g tetra(ethylene glycol), 1.02g tetra(ethylene glycol) dimethyl ether, and 0.018 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 1.75 g DVB, 1.98 g tetra(ethylene glycol), 1.02 gtetra(ethylene glycol) dimethyl ether, and 0.018 g AIBN.

Example 11

A column (50×4.6 mm i.d. stainless steel, which is 50 mm length and 4.6mm inner i.d.) was prepared as Example 1 with the followingpolymerization solution containing 1 g methyl methacrylate (MMA), 1 gEDMA, 1.8 g cyclohexanol, 1.2 g dodecanol and 0.02 g AIBN It wasconnected to a HPLC pump and washed with THF and water at 0.5 ml/min for20 minutes in sequence.

This column was subjected to a hydrolysis reaction as following: 2 ml 6mol/I NaOH was pumped through the column at the flow rate of 0.5 ml/min;The column was sealed by two column plugs and placed in a water bath at80?C for 1 hour. It was washed with 20 ml water at the flow rate of 0.5ml/min and characterized with protein separation and binding capacitymeasurement described in LC Characterization Method.

Alternative Versions of Example 11

A column was prepared as Example 11 with the following polymerizationsolution: 0.1 g acrylic acid (M), 0.9 g methyl methacrylate (MMA), 1 gEDMA, 3 g dodecanol and 0.02 g AIBN. The capacity of the column wasmeasured before and after hydrolysis. The capacity before hydrolysis wasabout 10 mg lysozyme per ml column volume. It was about 30 mg afterhydrolysis.

Another column was prepared as Example 11 with the followingpolymerization solution: 0.2 g AA, 0.8 g MMA, 1 g EDMA, 3 g dodecanoland 0.02 g AIBN. The capacity of the column was measured as incharacterization method 11. The capacity before hydrolysis was about 27mg lysozyme per ml column volume. It was about 50 mg after hydrolysis.

Another column was prepared as Example 11 with the followingpolymerization solution: 0.3 g AA, 0.7 g MMA, 1 g EDMA, 3 g dodecanoland 0.02 g AIBN. The capacity of the column was measured before andafter hydrolysis The capacity before hydrolysis was about 43 mg lysozymeper ml column volume. The capacity was more than 60 mg after hydrolysis.

Another column was prepared as above with the following polymerizationsolution: 0.4 g AA, 0.6 g MMA, 1 g EDMA, 3 g dodecanol and 0.02 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 0.1 g M, 0.9 g tert-butyl acrylate, 1 g EDMA, 3 g dodecanoland 0.02 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 0.3 g M, 0.3 g MMA, 1.4 g EDMA, 3 g dodecanol and 0.02 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 0.2 g M, 0.7 g MMA, 0.1 g HEMA, 1 g EDMA, 2.85 g dodecanol,0.15 g cyclohexanol and 0.02 g AIBN. Another column was prepared asabove with the following polymerization solution: 0.4 g M, 1.6 g DVB, 3g dodecanol and 0.02 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 1 g GMA, 1 g EDMA, 2.4 g cyclohexanol, 0.6 g dodecanol, 0.02 gAIBN. This column was subjected to an acid-catalyzed ring openingreaction as in a Alternative versions of Example 9 before base-catalyzedhydrolysis reaction as in Example 11.

Another column was prepared as above with the following polymerizationsolution: 0.5 g GMA, 0.5 g HEMA, 1 g EDMA, 1.8 g cyclohexanol, 1.2 gdodecanol, 0.02 g AIBN.

Another column was prepared as above with the following polymerizationsolution: 0.2 g M, 0.6 g MMA, 0.2 g GMA, 1 g EDMA, 3 g dodecanol and0.02 g AIBN. This column was subjected to an acid-catalyzed ring openingreaction before base-catalyzed hydrolysis reaction as above. It wasfurther hydrolyzed with the following solution: 0.25M Sodiumchloroacetate in 5M NaOH at 60° C. for 6 hours.

Another column was prepared as above with the following polymerizationsolution: 0.2 g M, 0.5 g MMA, 0.1 g GMA, 1.2 g EDMA, 2.55 g dodecanol,0.45 g cyclohexanol and 0.02 g AIBN. It was hydrolyzed by 0.25M Sodiumchloroacetate in 5M NaOH at 60?C for 6 hours.

All these columns were subjected to Bed Stabilization and CompressionMethod and characterized with protein separation and binding capacitymeasurement as in LC Characterization Method described above.

Example 12

A column (PEEK-lined stainless steel, 50×4.6 mm ID) was prepared asExample 1 with the following polymerization solution: 3 g GMA, 3 g EDMA,6.9 g cyclohexanol, 2.1 g dodecanol and 0.06 g AIBN.

This column was first modified with ring-opening reaction under acidiccondition. Five bed volume of the solution of 0.5 M sulfuric acid inwater was pumped through the columns. The column was sealed and heatedin a water bath at 50° C. for 4 hours. It was washed with 20 bed volumeof water after modification.

This column was further modified with a etherification reaction. Fivebed volume of a solution containing 20 g sodium chloroacetate, 20 g NaOHand 64 ml water was pumped through the column. The column was sealedwith column plugs and heated in a water bath at 60° C. for 2.5 hours. Itwas washed with water, stabilized and conditioned as Stabilization andConditioning Methods. This column was characterized as in LCCharacterization Method.

Alternative Versions of Example 12

The Example 12 was followed with different modification methods.

A column prepared as in Example 12 was modified by a ring-openingreaction with the following solution: 6 mol/I glycolic acid and 0.5 MTFI in water for 3 hours.

Another column was modified with the above ring-opening reaction andhydrolysis reaction in 5 M NaOH solution at 60° C. for 2.5 hours.

Another column was first modified by ring-opening reaction with thefollowing solution containing 40 g glycolic acid, 60 ml 0.5 Mtrifuoroacetic acid (TFA) for 2 hours. It was further modified with asolution containing 20 g ClCH₂COONa and 60 ml 5M NaOH for 3 hours.

Example 13

Thirty columns were prepared as Example 2 with the following solution:12 g AA, 30 g MMA, 6 g GMA, 72 g EDMA, 27 g cyclohexanol, 153 gdodecanol and 1.2 g AIBN. These columns were prepared by parallelsynthesis at the same time using three manifolds connecting to onesyringe pump to obtain 120 psi pressure during polymerization. Afterpolymerization, polymers were pushed out of the columns by a syringepiston (about 9 mm i.d.) for the following uses.

One polymer rod from above was trimmed to be smaller with the diameterabout 8 mm. It was cut to 1 cm thick discs. These discs were used asfillers for a second stage polymerization to prepare another column. 1.8ml solution was filled into a glass column (100×10 mm i.d.), one end ofwhich was sealed by pressurization device shown in FIG. 3. Six polymerdiscs were filled into the column one by one. All these discs should becovered by the solution. A Teflon stopper was used to seal the other endof the column. This pressurization device was connected to a syringepump which was used to add 120 psi pressure to polymerization solutionat constant pressure mode. The column was heated in a water bath at 60°C. for 20 hours. After polymerization, the column was taken out of thewater bath. The pressurization device was detached from the syringe pumpafter the pressure was released. The pressurization device was openedand slowly removed from the column while the column is still warm. Thiscolumn was washed with 20 bed volume of acetonitrile and water at theflow rate of 1 ml/min in sequence. It was stabilized and conditioned asin Stabilization and Conditioning Method. This column was modified with0.25 mol/I Sodium chloroacetate in 5M NaOH at 60° C. for 6 hours. It wascharacterized as in LC Characterization method.

Alternative Examples of Example 13

Another column (100 mm×35 mm ID, glass) was prepared with the two stagepolymerization method with the polymer rods as the fillers. This columnwas sealed with TEFLON plug in one end. The other end of the column wasconnected to N₂ tank. The polymerization was under 120psi for 20 hoursat 60° C.

Example 14

Eight short polymer rods (10 mm×34 mm ID) were prepared with thefollowing solution: 8 g acrylic acid, 20 g methyl methacrylate, 4 gglycidyl methacrylate, 48 g ethylene glycol dimethacrylate, 102 gdodecanol, 18 g cyclohexanol and 0.8 g AIBN. The polymer rods wereprepared under 120 psi N₂ pressure. The rods were used as fillers forthe preparation of a large diameter long column using the two stagepolymerization method as in Example 12. A glass column (100 mm×35 mm ID)was filled with the short columns and the same polymerization as above.One end of the column was sealed with a TEFLON plug and the other endwas connected with a N₂ tank. The polymerization was carried out under120 psi pressure at 60° C. for 20 hours. The column was washed with 20bed volume acetonitrile and water. It was subjected to hydrolysisreaction as following: 0.25M Sodium chloroacetate in 5M NaOH at 60?C for6 hours. The column was characterized as LC Characterization Methoddescribed above.

Example 15

A column (PEEK-lined stainless steel, 50 mm×4.6 mm ID) was prepared asin Example 1 with the following solution: 4 g GMA, 4 g EDMA, 2.8 gdodecanol, 9.2 g cycohexnanol and 0.08 g AIBN.

This column was first hydrolyzed by 1 M H₂SO₄ solution at 40° C. for 3hours. After hydrolysis, it was activated by pumping 5 bed volume of 5%sodium t-butoxide solution in DMSO through the column and heated in awater bath at 90° C. for 1 hour. Then it was modified with the solutioncontaining 20% of the activation solution and 80% of butane sultone at80° C. for 20 hours.

Alternative Versions of Example 15

A column was prepared as in Example 15 except that propane sultone wasused instead of butane sultone.

Another column was prepared as in Example 15 except the modification andactivation temperature was 120° C. instead of 90° C. in an oil bath.

Another column was prepared as in Example 15 with the followingsolution: 4 g HEMA, 4 g EDMA, 9.4 g dodecanol, 2.6 cyclohexanol and 0.08g AIBN. It was modified as in Example 15.

Another column was prepared as in Example 1 with the following solution:0.55 g GMA, 1.2 g EDMA, 0.25 g 2-Acrylamido-2-methyl-1-propanesulfonicacid (AMPS), 0.48 g NaOH, 0.5 g water, 1.86 g propanol, 0.64 gbutanediol and 0.02 g AIBN.

Another column was prepared by direct copolymerization of AMPS as abovebut was further modified with the modification method described inExample 15.

All these columns were characterized with the strong cation exchangeprotein separation and binding capacity measurement described in LCCharacterization Method.

Example 16

A column was prepared as Example 1 with the following polymerizationsolution: 45 ml tetramethoxysilane, 100 ml of 0.01 mol/l aqueous aceticacid, 9 g urea and 11.5 g poly(ethylene oxide) (MW 10000). This solutionwas prepared by stirring this mixture in ice bath for 30 minutes. Thepolymerization was carried out in the column under 600 psi pressure andat 40° C. for 24 hours. The column was then washed with 20 ml water atthe flow rate of 0.5 ml/min and pumped in 5 ml of 0.01 mol/I aqueousammonium hydroxide solution. The column was sealed and kept at 120° C.for 3 hours followed by ethanol wash.

Example 17

The inhibitors such as methyl ether hydroquinone or tert-butylcatecolwere removed from monomers by distillation or normal phasechromatography before uses.

A polymerization solution was prepared as Example 1, but with thefollowing polymerization solution: 240 mg p-terphenyl, 800 mg AIBN, 16 gstyrene and 16 g divinylbenzene (80%), 26.4 g mineral oil, and 21.6 g2-ethylhexanoic acid.

An empty glass column (10 mm inner i.d. and 100 mm length), one end ofwhich was sealed by a pressuring device shown in FIG. 4, was filled withthis solution until the column was full. A TEFLON-plug contained in thePEEK screw cap shown in the same figure, was used to seal the other endof the column. The polymerization was allowed to expose to x-ray with adosage of 600 R/hour for 72 hours at an x-ray tube voltage of 111 kVp.The obtained column was further heated to 70° C. for 2 hours. Then thecolumn was washed with hexane followed by hexane/acetone (50/50),acetone, acetonitrile, respectively with 20 bed volume of each solvent.

The device of FIG. 4 was detached from the syringe pump after thepressure was released. The device of FIG. 4 was opened and carefullyremoved from the column. It was found that the height of the polymer rodwas 4 mm shorter than the height of the polymerization solution insidethe column. The column was then fitted with the original HPLC columnfittings. The column was connected to a HPLC pump and washed withacetonitrile at 2 ml/min for 20 minutes at 45 degrees C.

The prepared column was further washed with 20 bed volume of water andcompressed with the piston to get rid of the void volume. The column wasthen characterized using LC characterization method described in 1a. Thechromatogram is attached in FIG. 9.

Alternative Versions of Example 17

Another column was prepared as in example 17 in a glass column (35 mmi.d.×100 mm length) with the following mixture: 30 g styrene, 30 gdivinylbenzene, 38.05 g mineral oil and 22.18 g 2-ethylhexanoic acid,0.518 g p-terphenyl and 1.31 g AIBN. The columns was washed with 20 bedvolume of acetonitrile and water respectively. It is characterized withLC characterization method la. The separation is shown in FIG. 9.

Another column was prepared as in example 17. The fittings from one endof the column were detached and the media was pressed out of the columnby pumping 10 ml/min acetonitrile into the column through the other end.The separation media was submitted for porosimetry studies using SEM andmercury porosimeter after drying in vaccum at 50° C. for 24 hours.

Another column was prepared as in example 17 using larger diameter glasscolumn (35 mm i.d.×100 mm length). The column was sealed with twoTEFLON-plug contained in the TEFLON screw caps instead of the device inFIG. 4. The polymer was pushed out of the column and dried as aboveexample. The polymer was submitted for SEM and porosimetry studies.

Another column was prepared as above example using large diameter column(35 mm×100 mm) but using the following polymerization solution: 240 mgp-terphenyl, 800 mg AIBN, 3.2139 g styrene, 28.8088 g divinylbenzene(80% pure), 37.4060 g 1-dodecanol, 13.25 g toluene.

Another column was prepared as above example using large diameter column(35 mm×100 mm) but using the following polymerization solution: 240 mgp-terphenyl and 800 mg AIBN was dissolved in the monomer mixture of32.0034 g divinylbenzene (80%). Into the monomer mixture, 31.6926 gtetraethylene glycol, 16.3224 g tetraethylene glycol dimethyl ester wasadded.

Another column was prepared as above example within a polymeric housingdescribed in FIGS. 12-15. The polymerization solution contains 73.2 gdivinylbenzene, 73.4 g styrene, 85.2 g mineral oil, 60.8 g2-ethylhexanoic acid, 0.882 g p-terphenyl and 2.94 g AIBN. Thetemperature of polymerization in the center of the column was about thesame as the one on the edge of the column. The conversions of monomerswere almost complete after 4 days of 110 kV x-ray irradiatedpolymerization. Complete reaction is attained thermally as the furtherexotherm has no significant bad effect.

Many other columns using different scintillators, photo initiators,monomers and porogenic solvents have been prepared. The porogenicsolvents used include other alkane such as octane, alcohols suchmethanol, propanol and cyclohexanol, ethers such as. tetrahydrofuran,dioxane, oligomers such tetraethylene glycol, tetraethylene glycoldimethyl ether. Photo initiators used include 2-chlorothioxanthen-9-one,4,4′-bis(dimethylamino)benzophenone, 4,4′-bis(diethylamino)benzophenone,phenanthrenequinone, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide andazo bisisobutylronitrile (AIBN). The scintillators used include2,5-diphenyloxazole (PPO), 2-phenyl-5-(4-biphenylyl ) 1,3,4-oxadiazole(PBD), 2-(1-Naphthyl)-5-phenyloxazole (á-NPO) besides p-terphenyl andZnSe. The monomers used include acrylonitrile, butyl methacrylatebesides glycidyl methacrylate, ethylene glycol dimethacrylate, styrene,divinylbenzene, ethyl styrene.

In FIG. 9, there is shown a chromatogram of a separation in twodifferent diameter columns of the mixture of (1) Met-Enkephalin, (2)Leu-Enkephalin, (3) Angiotensin, (4) Phyusalaemin and (5) Substances Pon Poly (DVB-co-St) monolithic columns prepared by X-ray irradiationinitiated polymerization in column with a diameter of 10 mm and 35 mmand a length of 65 mm; Mobile phase: (A) water with 0.15% (v/v) TFA;Gradient: 10-40% B in A in 7 bed volume at a flow rate of 5 ml/min I.D.column and 50 ml/min for 35 I.D. column: Detection: UV at 214 nm.

In FIG. 10, there is shown a top view of an ultraviolet or visible lightpolymerization apparatus 150 having a stationary top surface 152, arotating top surface 156, a support member 157 connected to thestationary support surface 152 and pinned to the rotating surface 156 topermit rotation thereof and four fluorescent lamp holders 154A-154D.Visible or ultraviolet fluorescent lamps are inserted in these holders.

In FIG. 11, there is shown a schematic representation of a sidesectional view of the polymerization apparatus 150 showing the drivenmember 156 rotated reciprocally by a motor 159 to rotate thepolymerization apparatus 162. Two of the four lamps 166D and 166B areshown mounted to the lamp holders 154D and 154B and correspondingholders at the bottom end of the elongated lamps 166D and 166B. A piston164 is used to pressurize the polymerization mixture at 162 duringpolymerization. A fan 158 aids in cooling the polymerization apparatusand reflective coatings on the cover, the sides and the lamps reflectlight back into the light conducting walls of the container 162. Withthis arrangement, the lamps 166A-166D cause light to impinge on thepolymerization mixture to initiate and control the polymerizationreaction. The polymerization of relatively large diameter columns may beperformed while maintaining radial uniformity in the final plug at alllocations along the column in the direction of flow of the solvent andanalyte. The light may be turned on and off as desired to control thetemperature gradients so that the polymerization may take place under acombination of light and temperature in a controlled manner foruniformity.

In FIG. 12, there is shown a simplified elevational view of an apparatus170 for polymerization using principally x-ray radiation having aradiation proof cabinet 172, with a door 174, an upper window 176, aholder 178 and a container 180 to contain a reaction mixture at 182. Apiston 184 may be utilized in some embodiments to pressurize thereaction mixture 182. In the apparatus 170, x-rays or other suitableradiation such as gamma rays may be used to control the reaction in thepolymerization container in a safe convenient manner.

In some embodiments, pressure may be applied through the piston 184 byapplying air through the conduit 186 to move the piston inwardly againstthe reaction mixture 182 in a manner described above in connection withother embodiments. In the preferred embodiment, the apparatus 170 is asmall user friendly cabinet x-ray system resembling a microwave in thatit has a door and controls mounted on the cabinet. It uses low voltagelevels and can be operated by personnel safely from next to the cabinetbecause it has low penetration which is sufficient however for largecolumns. It is suitable for the polymerization of this invention becauseprocesses described hereinabove use added substances to aid inpolymerization such as photo initiators, fluorescing solvents, orporogens, x-ray sensitizers and/or scintillators. This unit permitsx-ray control of the polymerization and other units such as those ofFIG. 10 and 11 permit other radiation control of polymerization, thuspermitting for example control of polymerization with the aid ofradiation up to a point and finishing the polymerization using heat todecrease the time and yet avoid destructive head build-up.

In FIG. 13, there is shown a top view of the reaction vessel 180 havinga reactant entry opening 200, a coolant fluid inlet 203, a coolantoutlet 205, a casing 204 and an overflow outlet 202. The coolant ispreferably water. An opening 206 for air to move the plug 182 (FIG. 14)against the polymerization mixture AT 212 for pressure thereon isprovided. A thermocoouple can be provided through the opening 200 afterthe reactant mixture is place by turning over the vessel 180 and a plugcan be inserted there as well. With this arrangement, the reactionmixture may be irradiated under pressure if desired and subjected tox-rays axially for initiation and control of the polymerization. Waterflows through it as a coolant so that the combination of radiation andpressure can control thermal gradients and promote uniformity in thefinal chromatography plug or support.

In FIG. 14, there is shown a sectional view taken through section lines14-14 of FIG. 13 showing the transparent x-ray radiation window 192, theport 202 for the coolant water, the opening 200 for reactant, athermocouple 215 and its conductor 218 and a pin in that sequence, theair pressure opening 206 to move the plug 182 to pressurize the reactionmixture in 212, the reactant housing at 212 for receiving reactantmixture, a first distribution plate at 213 to distribute the solvent andanalyte during chromatography when the vessel 180 is used as a columnand the second distributor plate 215 to receive the solvent and analyteafter separation if the column at 212 after polymerization. With thisarrangement, radiation passes through the window 192 to control thepolymerization of the reactant 182. This may be done under pressureapplied by the plug 182.

In FIG. 15, there is shown a sectional view of the polymerization vessel180 taken through lines 15-15 of FIG. 13 showing a piston 182, an airspace 210 applying pressure to the plug 182 to pressurize the reactantat 212 while it is being irradiated by x-rays and cooled by flowingwater in reservoir 190.

As can be understood from the above description, a polymerizationmixture which may include a porogen or solvent is polymerized into aporous plug under the control of radiation. For this purpose, there isat least one substance that is caused by the radiation to effectpolymerization. Some of the substances may emit radiation that effectother substances which in turn initiate or promote polymerization. Forthis purpose, the radiation mixture should include at least one monomer,at least a porogen or a solvent and a substance that effectspolymerization. X-rays may be used with only a monomer to prepare asupport. If a porogen or solvent is included, the support may be porous.The x-rays are a particularly safe type of radiation and may have wideapplication in forming polymeric supports. The radiation may causepolymerization or irradiate a substance such as a solvent that emitsfurther energy that causes initiation or promotion of polymerization.

Example 18

Homopolymers, polymer resins and porous polymer support have beenprepared using 110 kV x-ray irradiation. The polymers were prepared inthe glass vials and columns.

A polymerization mixture containing 1% AIBN and 0.1% p-terphenyl instyrene was degassed as described in the degassing method and filledinto and 1 ml glass vial. The vial was sealed with a screw cap. The vialwas exposed in 600 R/hour x-ray using 111 kVp power for two days. Arigid bulk polystyrene polymer was obtained in the shape of the vialafter breaking the glass of the vial.

Alternative Versions of Example 18

A homopolyglycidyl methacrylate was prepared according to the abovemethod.

A homopolystyrene was prepared using solution polymerization accordingto the above method. 50% of styene in toluene containing 1% of AIBN and0.1% of p-terphenyl was polymerized under 600 R/h x-ray for two days.Polystyrene was obtained after polymerization.

A poly(styrene-co-divinylbenzene) resin was prepared according to theabove method using the following 1:1 ratio of styrene and divinylbenzene. The polymerization mixture contains 0.9 g styrene, 0.9 gdivinylbenze, 17 mg AIBN, 5.7 mg p-terphenyl. The polymer resin wasobtained after polymerization. Gelation happened after 5 hours ofpolymerization.

A poly(glycidyl-co-ethylene glycol dimethacrylate) resin was preparedaccording to the above method using the following 1:1 ratio of glycidylmethacrylate (GMA) and ethylene glycol dimethacrylate (EDMA). Thepolymerization mixture contains 0.9 g GMA, 0.9 g EDMA, 17 mg AIBN, 5.7mg p-terphenyl. The polymer resin was obtained after polymerization.Gelation happened after 5 hours of polymerization.

A poly(glycidyl-co-ethylene glycol dimethacrylate) porous support wasprepared according to the above method using the following 1:1 ratio ofglycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA).The polymerization mixture contains 0.45 g GMA, 0.46 g EDMA, 0.91 gcyclohexanol, 20 mg AIBN, 1.5 mg p-terphenyl. A porous polymer wasobtained after polymerization. Gelation happened after 6 hours ofpolymerization.

A poly(styrene-co-divinylbenzene) porous support was prepared accordingto the above method using the following polymerization mixture contains0.45 g styene, 0.45 g divinylbenzene (80% pure), 0.91 g cyclohexanol, 20mg AIBN, 6.5 mg p-terphenyl. A porous polymer was obtained afterpolymerization. Gelation happened after 4.5 hour of polymerization.

Example 19

Silica capillary with different innerdiameters including 75 μm, 100 μm,200 μm, 250 μm, 320 μm, 530 μm and 700 μm were modified with 1 M sodiumhydroxide solution at 90° C. for 2 hours in an oven. The capillary wasthen washed with 60 column volumes of deionized water and acetone. Itwas dried by nitrogen purging through the column for 20 minutes. Thecapillary tubing was filled with a silanizing solution containing 50%(v/v) 3-(trimethoxysilyl)propyl methacrylate and 0.02% (w/v)hydroquinone in N,N-dimethylformamide (DMF). After both ends of thecapillary were sealed, it was heated in an oven at 100° C. for about 10h, and then washed with DMF and acetone. The capillary was dried with anitrogen purging after wash.

Polymerization solutions were prepared as described in Example 1 withthe composition of components listed in Table 1.

Each modified capillary (usually 15˜20 cm) was filled with abovepolymerization solution. Two ends of the capillary were sealed in two1.8 mL glass vials, respectively, which were also filled withpolymerization solution. Teflon and parafilm were used to double sealthe cap of the vial. About 500 μL empty space was left in each vial andthe ends of the capillary locate at the half height of the vial. Two tofour columns can be prepared by using the same sealing vials. Thecapillaries were hung vertically in the water bath at certaintemperature listed in Table 1 for 20 hours by clamping the top vial on astand. After polymerization, the monolithic capillary columns werewashed with about 20 column volume organic solvent, usuallyacetonitrile, sometimes hexane when mineral oil was used as porogen.

The columns were characterized with the LC characterization method 1a atthe flow rate of 3, 5 and 10 μl/min. Great resolutions have beenachieved. TABLE 1 Polymerization solution composition and polymerizationconditions. I II III IV V vol vol wt vol wt Reactants % g % g % g % g %g Styrene 20 2.1 16 1.6 20 2.0 20 2.1 8 0.8 Divinylbenzene 20 2.1 24 2.520 2.0 20 2.1 32 3.2 Tetrahydro- 7.5 0.8 furan chloro- 9.5 1.1cyclohexane THP 7.0 0.7 1-decanol 52.5 5.0 50.5 4.8 53 5.1 Toluene 8 0.81-dodecanol 52 5.2 1- 27 2.7 ethylhexanoic acid Mineral oil 33 3.3 AIBN0.04 g Polymerization 70 70 70 70 80 Temp, ° C.

Alternative Versions of Example 19

A monolithic capillary column was prepared as in the above example withthe following polymerization solution: 0.40 g BMA, 2.6 g EDMA, 3.8 g1-propanol, 1.6 g 1,4-butanediol, 0.6 g water and 0.04 g AIBN. Thepolymerization was carried out at 60° C. This column was characterizedwith the LC characterization method 1 a with a microanalytical liquidchromatography system. The flow rate was 5 μl/min. Excellent separationsof proteins were achieved.

Many other poly(acrylate) based capillary monoliths were synthesizedwith the variation of BMA /EDMA ratios (1/3 to 3/2) and differentporogen concentrations (1-propanol with 34 to 39 wt % of the totalreaction mixture while 1,4-butanediol with 20 to 15 wt %).

Another column was prepared as in Example 19 with the followingpolymerization mixture: 2.2 g DVB, 1.3 g styrene, 0.9 g acrylonitrile,4.7 g mineral oil, 0.8 g toluene and 0.044 g AIBN. The polymerizationtemperature is 75° C. Many other columns of this type were prepared withthe variation of acrylonitrile content in the polymer matrix from 0 to50%. The polymers showed increasing hydrophilicity butdifferentretentativitiesof proteins.

Another column was prepared as in Example 19 with the followingpolymerization mixture: 0.8 g GMA, 2.4 g EDMA, 0.8 g ATMS, 3.1 g1-propanol, 2.6 g 1,4-butanediol, 0.3 g water and 0.04 g AIBN. Thepolymerization reaction was carried out at 60° C. for 20 hours. Afterpolymerization, the porogenic solvents in columns were washed away withaceonitrile. Then they were filled with modification solution, which isa mixture of TMA and water with volume ratio of 1 to 2 and with 0.45g/mL TMA HCl. The modification was carried out at 40° C. for 4 hours.

Example 20

The polymerization solution is prepared as in Example 1 but with thefollowing mixure: 3.2154 g acrylic acid, 8.0026 g methyl methacrylate,1.6064 g glycidyl methacrylate, 19.2055 g ethylene glycoldimethacrylate, 43.8952 g 1-dodecanol, 9.0017 g cyclohexanol, 320 mgdiphenyl (2,4,6 trimethyl(benzole) phosphine oxide. The polymerizationmixture was sonicated for 5 minutes and poured into the column, one endof which was sealed by a TEFLON cap, then the column was sealed withanother TEFLON cap. The polymerization was allowed to expose to ordinaryceiling light for 7 days. The column was washed with 20 bed volume ofacetonitrile followed by 20 bed volume of water. The column wascharacterized with chromatography.

Alternative Versions of Example 20

A glass column of size 10 cm×1 cm ID was prepared as the above examplewith the following mixture: 2.5074 g glycidyl methacrylate, 2.5003 gethylene glycol dimethacrylate, 7.5003 g p-xylene, and 58.2 mg diphenyl(2,4,6 trimethyl(benzole) phosphine oxide. The polymerization mixturewas exposed to ordinary ceiling light for 24 hours. The samepolymerization was carried out in another 2 ml vial for 24 hours. Theconversion of monomers was 92%.

A homopolyglycidyl methacrylate was prepared in a vial with thefollowing polymerization mixture: 10 g GMA and 0.1 g diphenyl (2,4,6trimethyl(benzole) phosphine oxide. The solution was exposed to ordinaryceiling light for 24 hours. The polymer gelled up after 3 hours. Veryclear polymer was obtained.

A homopoly(glycidyl methacrylate) was prepared in a vial with followingpolymerization mixture: 10 g GMA, 10 g xylene and 0.1 g diphenyl (2,4,6trimethyl(benzole) phosphine oxide. The solution was exposed to ordinaryceiling light for 24 hours. The polymer gelled up after 3 hours. Clearpolymer was obtained.

From the above description it can be understood that the novelmonolithic solid support of this invention has several advantages, suchas for example: (1) it provides chromatograms in a manner superior tothe prior art; (2) it can be made simply and inexpensively; (3) itprovides higher flow rates for some separations than the prior artseparations, thus reducing the time of some separations; (4) it provideshigh resolution separations for some separation processes at lowerpressures than some prior art processes; (5) it provides high resolutionwith disposable columns by reducing the cost of the columns; (6) itpermits column of many different shapes to be easily prepared, such asfor example annular columns for annular chromatography and prepared inany dimensions especially small dimensions such as for microchips andcapillaries and for mass spectroscopy injectors using monolithicpermeable polymeric tips; (7) it separates both small and largemolecules rapidly; (8) it can provide a superior separating medium formany processes including among others extraction, chromatography,electrophoresis, supercritical fluid chromatography and solid supportfor catalysis, TLC and integrated CEC separations or chemical reaction;(9) it can provide better characteristics to certain known permeablemonolithic separating media; (10) it provides a novel approach for thepreparation of large diameter columns with homogeneousseparation-effective opening size distribution; (11) it provides aseparation media with no wall effect in highly aqueous mobile phase andwith improved column efficiency: (11) it improves separation effectivefactors; and (12) it reduces the problems of swelling and shrinking inreverse phase columns.

Although preferred embodiments of the inventions have been describedwith some particularity, many variations in the invention are possiblewithin the light of the above teachings. Therefore, it is to beunderstood that, within the scope of the appended claims, the inventionmay be practiced other than as specifically described.

1-18. (canceled)
 19. A method of performing chromatography comprisingthe steps of: applying a sample to a chromatographic column havingcolumn casing and a size compensated polymer monolithic packing; andsupplying a solvent to the chromatographic column, whereby the sample isseparated into its components within the chromatographic column. 20-30.(canceled)
 31. A chromatographic column comprising: a chromatographiccolumn support having internal column walls; a monolithic permeable plugin said column walls; said permeable monolithic polymeric plug being apolymer having separation-effective openings of a controlled size formedin the polymer by a porogen in the polymerization mixture beforepolymerization and controlled in size at least partly by pressure duringpolymerization.
 32. A chromatographic column in accordance with claim 31in which the permeable monolithic polymeric plug has smooth walls.
 33. Achromatographic column in accordance with claim 31 wherein there aresubstantially no pores within the permeable monolithic polymeric plug.34. A chromatographic column in accordance with claim 31 wherein thepermeable monolithic polymeric plug is free of channeling openings inthe walls of the plug.
 35. A chromatographic column in accordance withclaim 31 wherein the permeable monolithic polymeric plug is formedprincipally of methacrylate
 36. A chromatographic column in accordancewith claim 31 wherein the permeable monolithic polymeric plug is formedprincipally of methacrylate with hydrophobic surface groups. 37-38.(canceled)
 39. A chromatographic column in accordance with claim 31wherein the permeable monolithic polymeric plug is principally formed ofpolymers of glycidyl methacrylate and of ethylene dimethacrylate in aratio by weight in the range of from 1 to 1 and 2 to
 1. 40. (canceled)41. A column in accordance with claim 31 in which the permeablemonolithic polymeric plug includes as its principal component a mixtureof divinylbenzene and styrene. 42-44. (canceled)
 45. A weak ion exchangechromatographic column comprising: a chromatographic column supporthaving internal column walls; a permeable monolithic polymeric plug insaid column walls; said permeable monolithic polymeric plug being formedprincipally of methacrylate polymer and having smooth walls.
 46. A weakion exchange chromatographic column comprising: a chromatographic columnsupport having internal column walls; a permeable monolithic polymericplug in said column walls; said permeable monolithic polymeric plugbeing formed principally of polymers of glycidyl methacrylate and ofethylene dimethacrylate in a ratio by weight in the range of from 1 to 1and 2 to
 1. 47. (canceled)
 48. A chromatographic column comprising: achromatographic column support having internal column walls; a permeablemonolithic polymeric plug in said column walls; said permeablemonolithic polymeric plug being formed principally of a mixture ofdivinylbenzene and styrene and having smooth walls. 49-58. (canceled)59. Apparatus for making a chromatographic column comprising: atemperature controlled reaction chamber adapted to contain apolymerization mixture during polymerization; and means for applyingpressure to said polymerization mixture in said temperature controlledreaction chamber.
 60. (canceled)
 61. Apparatus according to claim 59wherein said means for applying pressure is a mean for applying pressurewith a movable member.
 62. Apparatus according to claim 59 in which saidmovable member has a smooth surface positioned to contact saidpolymerization mixture as pressure is applied during polymerization. 63.Apparatus for making a chromatographic column comprising: a temperaturecontrolled reaction chamber adapted to contain a polymerization mixtureduring polymerization to form a plug; aqueous processing means forapplying an aqueous solution to the plug; means for applying pressure tosaid plug to reduce voids in the plug.
 64. A method of making amonolithic chromatographic column comprising the steps of: preparing apolymerization mixture; performing polymerization under pressure,whereby sufficient pressure is applied to prevent voids from beingformed caused by vacuum due to shrinkage during polymerization.
 65. Themethod of claim 64 in which the step of performing polymerizationincludes the step of inserting a polymerization mixture into the wallsof a chromatographic column and polymerizing in a temperature controlledchamber during at least part of the time pressure is being applied tothe polymerization mixture.
 66. The method of claim 65 in which the stepof inserting a polymerization mixture includes the step of inserting atleast one vinyl monomer, an initiator, and a porogen.
 67. A method ofmaking a monolithic chromatographic column comprising the steps of:adding a polymerization mixture containing a porogen to a closedcontainer; polymerizing the mixture under pressure to form a polymerplug, whereby sufficient pressure is applied to prevent voids from beingformed caused by vacuum due to shrinkage during polymerization; andwashing the polymer plug to remove the porogen.
 68. A method of making amonolithic chromatographic column comprising the steps of: preparing apolymerization mixture including a porogen; performing polymerization toform a polymer plug; washing the polymer plug to remove the porogen,wherein the plug tends to swell; and applying pressure to preventswelling of the polymer plug and eliminate voids.
 69. A method of makinga monolithic permeable device for separating the components of a sample,comprising the steps of: preparing a polymerization mixture including amonomer and a porogen wherein the porogen includes a mixture ofalcohols. 70-93. (canceled)
 94. A method of controlling polymerizationof a porous medium comprising the steps of: establishing apolymerization mixture including a porogen; controlling the rate of thepolymerization with radiation and at least one substance that is causedby the radiation to affect the polymerization. 95-100. (canceled)
 101. Amethod of making a monolithic permeable device for separating thecomponents of a sample, comprising the steps of: preparing apolymerization mixture including a monomer, a porogen and at least oneradiation sensitive substance; said radiation sensitive substancecomprising one or more substances selected from a group consisting ofphoto initiators, x-ray sensitizers and scintillators; and irradiatingthe polymerization mixture.
 102. A method of making a monolithicpermeable plug, said method comprising polymerizing a mixture to form asolid support containing at least one monomer and a porogen comprisingthe step of irradiating the mixture with X rays. 103-104. (canceled)105. An apparatus for polymerizing chromatographic columns comprising: atemperature controlled reaction chamber adapted to contain apolymerization mixture during polymerization to form a monolithicpermeable plug; radiation means for applying radiation to the plug; andcontrol means for controlling the radiation.
 106. An apparatus inaccordance with claim 105 further including means for applying pressureto the plug to reduce voids in the plug.